Table 1.
Comparison of germination and growth of strains of Aspergillus spp. studied on Sabouraud agar and on PAO placed on Sabouraud plates.
Table 2.
Comparison of Echinocandin susceptibility of Aspergillus spp. studied on Sabouraud agar and on PAO placed on Sabouraud plates.
Figure 1.
Imaging microcolonies of A. fumigatus JBZ11 grown on PAO with echinocandins by SEM.
Culture was for 14 h at 37°C on Sabouraud medium with imaging from directly above unless stated otherwise. A: Microcolony without drugs. B: Growth with 0.0625 µg/ml anidulafungin. Imaging from the side at a 30 degree angle. C: Growth with 0.06 µg/ml anidulafungin. D: Growth with 2 µg/ml anidulafungin. E: Growth with 0.5 µg/ml caspofungin. Arrows indicate lysed hyphal tips. Scale bar in panel A indicates 90 µm. When applied to panel B, 8 µm; panel C, 30 µm; panel D, 15 µm; panel E, 30 µm.
Figure 2.
Growth of microcolonies of A. fumigatus grown on PAO with echinocandins.
A: Microcolony growth from conidia of sensitive strain JBZ11 without drugs (solid squares), with 0.125 µg/ml anidulafungin (open squares) or 1 µg/ml anidulafungin (solid triangles), with 0.125 µg/ml caspofungin (open triangles) or 1 µg/ml caspofungin (solid circles) or 0.1 µg/ml amphotericin B (solid diamonds). B: As panel A, except using caspofungin resistant and anidulafungin sensitive strain CWZ1243.
Figure 3.
Lysis of microcolonies of A. fumigatus JBZ11 grown on PAO with echinocandins.
Culture was for 14 h at 37°C. A: Microcolony cultured on 0.125 µg/ml anidulafungin. Staining was with Fun-1/calcofluor white and imaging by fluorescence microscopy. B: Imaging by SEM with vapour fixation of a hyphal tip grown without drugs. C: As panel B except growth with 0.125 µg/ml anidulafungin. D: As panel C, except growth with 0.125 µg/ml caspofungin. Arrows indicate lysed hyphal tips. Scale bar in panel A indicates 20 µm. When applied to panel B, 3 µm; panel C, 4 µm; panel D, 4 µm.
Figure 4.
Examples of imaging microcolonies of Aspergillus spp. grown on PAO with 0.5 µg/ml caspofungin and dual stained with propidium iodide and Syto9.
Culture in all cases was for 14 h at 37°C on Sabouraud medium. Panels A–C; CASP sensitive strain of A. fumigatus (JBZ11). A: Staining with PI. B: Staining with Syto9. C: Merged. Arrows in panel A indicate lysed hyphal tips. Panels D–E; as previous panels but imaging an CASP resistant strain of A. fumigatus (CWZ1243) showing greater growth and only limited echinocandin mediated damage (e.g. arrow). G–I; as panels A–C, but showing staining of an ANI and CASP sensitive strain of A. terreus (CWZ59). Scale bar in panel C indicates 20 µm for panels A–C, 45 µm for panels D–F and 15 µm for G–I.
Figure 5.
Quantification of the lysis of hyphal tips by echinocandins.
Panels A and B: Echinocandin susceptible strain of A. fumigatus JBZ11. C and D: Caspofungin resistant, anidulafungin sensitive strain of A. fumigatus CWZ1243. Culture was for 14 h at 37°C on Sabouraud medium. A and C: Effect of anidulafungin. Black, average number of intact hyphal tips per microcolony. White, average number of lysed hyphal tips per microcolony. B and D: Effect of caspofungin. Interpretation as panel A.
Figure 6.
Quantification of the lysis of hyphal tips by echinocandins by sample preparation method.
Effect of sample preparation method on percentage lysed hyphal tips. Black bars, 0.125 µg/mlanidulafungin. White bars, 0.5 µg/ml caspofungin. FCS, Fun-1 and calcofluor white staining on Sabouraud medium with imaging by fluorescence microscopy. FCOS, Fun-1 and calcofluor white staining with osmotic shock on water agar and fluorescence microscopy. SPS, Syto9 and propidium iodide staining on Sabouraud medium with assessment by fluorescence microscopy. SPOS, Syto9 and propidium iodide staining with osmotic shock on water agar and fluorescence microscopy. SEMGEL, glutaraldehyde fixation by gel method and imaging by SEM. SEMVAP vapour fixation method and SEM imaging. Error bars indicate S.D. from the mean (n = 3).
Figure 7.
Recovery from echinocandins by sensitive strain JBZ11.
A: Recovery from anidulafungin. Open squares, initial growth on 0.125 µg/ml anidulafungin then shift to Sabouraud agar with no drugs at 14 h (indicated by arrow). Open triangles, downshift from 1 µg/ml anidulafungin to none at 14 h. Solid squares, control with growth on 0.125 µg/ml throughout experiment. Solid triangles, continuous growth on 0.125 µg/ml anidulafungin. B: Recovery from caspofungin. Open squares, initial growth on 0.125 µg/ml caspofungin then shift to Sabouraud agar with no drugs at 14 h (indicated by arrow). Open triangles, downshift from 1 µg/ml caspofungin to none at 14 h. Solid squares, control with growth on 0.125 µg/ml throughout the experiment. Solid triangles, continuous growth on 0.125 µg/ml caspofungin.
Figure 8.
Effect of combining cyclosporine A with anidulofungin or caspofungin on the growth of A. fumigatus JBZ11.
The average microcolony diameter (+/− S.D.) was determined from >100 microcolonies/data point after 16 h culture on PAO placed on Sabouraud medium containing the drugs. Drug concentrations are given in µg/ml.