Synergy, pharmacodynamics, and time-sequenced ultrastructural changes of the interaction between nikkomycin Z and the echinocandin FK463 against Aspergillus fumigatus

We investigated the potential synergy between two cell wall-active agents, the echinocandin FK463 (FK) and the chitin synthase inhibitor nikkomycin Z (NZ), against 16 isolates of filamentous fungi. Susceptibility testing was performed with a broth macrodilution procedure by NCCLS methods. The median minimal effective concentration (MEC) of FK against all Aspergillus specie s was 0.25 mug/ml (range, 0.05 to 0.5 mug/ml). For Fusarium solani and Rhiz opus oryzae, MECs of FK were > 512 mug/ml. The median MEC of NZ against Asp ergillus fumigatus was 32 mug/ml (range, 8 to 64 mug/ml), and that against R. oryzae was 0.5 mug/ml (range, 0.06 to 2 mug/ml); however, for the other Aspergillus species, as well as F. solani, MECs were > 512 mug/ml. A checke rboard inhibitory assay demonstrated synergy against A. fumigatus (median f ractional inhibitory concentration index = 0.312 [range, 0.15 to 0.475]). T he effect was additive to indifferent against R. oryzae and indifferent aga inst other Aspergillus spp. and F. solani. We further investigated the phar macodynamics of hyphal damage by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diph enyltetrazolium bromide] assay and examined the time-sequenced changes in h yphal ultrastructure. Significant synergistic hyphal damage was demonstrate d with the combination of NZ (2 to 32 mug/ml) and FK (0.03 to 0.5 mug/ml) o ver a wide range of concentrations (P < 0.001). The synergistic effect was most pronounced after 12 h of incubation and was sustained through 24 h. Ti me-sequenced light and electron microscopic studies demonstrated that struc tural alterations of hyphae were profound, with marked transformation of hy phae to blastospore-like structures, in the presence of FK plus NZ, while f ungi treated with a single drug showed partial recovery at 24 h. The method s used in this study may be applicable to elucidating the activity and inte raction of other cell wall-active agents. In summary, these two cell wall-t argeted antifungal agents, FK and NZ, showed marked time-dependent in vitro synergistic activity against A. fumigatus.