Polymyxin B, in combination with fluconazole, exerts a potent fungicidal effect

Abstract

Objectives: The objective of this study was to identify existing clinical compounds that either possess a fungicidal activity alone or can act synergistically with fungistatic antifungals.

Methods: We screened a clinical compound library for drugs that exhibited anti-Aspergillus activity. Among selected compounds, the cationic peptide antibiotic polymyxin B was chosen for further characterization because it can be used parenterally and topically. The fungicidal effect of polymyxin B and its synergistic interactions with azole antifungals were tested against a variety of fungal species. The toxicity of the drug combination of polymyxin B and fluconazole was compared with that of each drug alone in mammalian cell cultures.

Results: We found that polymyxin B possesses a broad-spectrum antifungal activity at relatively high concentrations. However, because of its synergistic interactions with azole antifungals, polymyxin B at much lower concentrations exerts a potent fungicidal effect against Cryptococcus neoformans, Candida albicans and non-albicans Candida species and moulds when combined with azoles. The combination of polymyxin B and fluconazole at concentrations within susceptible breakpoints is particularly potent against C. neoformans isolates, including fluconazole-resistant strains. The drug combination displayed no additional toxicity compared with polymyxin B alone when tested in cell culture.

Conclusions: The combination of polymyxin B and fluconazole has the potential to be used in the clinic to treat systemic cryptococcosis. Our findings suggest that combining cationic peptide antibiotics with azole antifungals could provide a new direction for developing novel antifungal therapies.

Figure 1

Polymyxin B is fungicidal against Aspergillus fumigatus. (a) A. fumigatus spores germinated and formed long hyphae after overnight growth in drug-free medium. (b) Polymyxin B at 20 µM (28 mg/L) final concentration in Y + UU liquid medium inhibited germination of the majority of A. fumigatus spores. (c) A. fumigatus conidia transferred to drug-free medium after an overnight treatment with 20 µM polymyxin B did not germinate after 24 h of incubation at 37°C. (d) Pre-germinated germ tubes of A. fumigatus did not grow in the presence of 20 µM polymyxin B. In this experiment, germ tubes were formed after a 7 h incubation of spores in a drug-free medium, and then subjected to an overnight treatment with 20 µM polymyxin B, and subsequently followed by an incubation in drug-free medium for 24 h. Note that the germ tubes did not become longer hyphae, in contrast to the non-treated cells shown in (a). (e) Quantitative analysis of the effectiveness of polymyxin B at 20 µM (28 mg/L) or 40 µM (56 mg/L) in inhibiting the germination of A. fumigatus spores. Spores of A. fumigatus strain B5233 were incubated at 37°C for 9.5 h. One hundred and fifty cells were counted for each treatment. The averages and standard deviations based on three independent experiments are shown in the graph.

Figure 2

Synergistic interaction between fluconazole and polymyxin B against Candida and Cryptococcus. Discs containing water, polymyxin B, fluconazole and the drug combination were dried and overlaid on a lawn of yeast cells derived from the strains indicated. Cells were incubated for 24 h (Candida species) or 48 h (Cryptococcus). Inhibition of fungal growth in regions surrounding the disc produces a halo. A completely clear halo indicates fungicidal activity. (a) Disc diffusion halo assays of the C. albicans strain SC5314 produced a clear halo when fluconazole and polymyxin B were used in combination. (b) Microscopic observations reveal significant clearing of the zone of inhibition (halo) when fluconazole and polymyxin B were used in combination. The left-hand side of each image shows the edge of the disc. PMB, polymyxin B; FLC, fluconazole.

Figure 3

The combination does not produce any additional adverse effect on host cells. The growth of human monocytic THP-1 cells was not affected by the combination of polymyxin B sulphate and fluconazole at final concentrations of 40 and 100 mg/L, respectively. For all three timepoints, there are no statistical differences based on Student's t-tests between the control and treatments, with P values no greater than 0.01.