Quantification of the lysis of hyphal tips by echinocandins.

<p>Panels A and B: Echinocandin susceptible strain of <i>A. fumigatus</i> JBZ11. C and D: Caspofungin resistant, anidulafungin sensitive strain of <i>A. fumigatus</i> 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.</p

Lysis of microcolonies of <i>A. fumigatus</i> JBZ11 grown on PAO with echinocandins.

<p>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.</p

Comparison of Echinocandin susceptibility of <i>Aspergillus</i> spp. studied on Sabouraud agar and on PAO placed on Sabouraud plates.

a<p>MIC values in µg/ml.</p>b<p>MEC values in µg/ml.</p

Imaging microcolonies of <i>A. fumigatus</i> JBZ11 grown on PAO with echinocandins by SEM.

<p>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.</p

Recovery from echinocandins by sensitive strain JBZ11.

<p>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.</p

Effect of combining cyclosporine A with anidulofungin or caspofungin on the growth of <i>A. fumigatus</i> JBZ11.

<p>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.</p

Examples of imaging microcolonies of <i>Aspergillus spp.</i> grown on PAO with 0.5 µg/ml caspofungin and dual stained with propidium iodide and Syto9.

<p>Culture in all cases was for 14 h at 37°C on Sabouraud medium. Panels A–C; CASP sensitive strain of <i>A. fumigatus</i> (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 <i>A. fumigatus</i> (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 <i>A. terreus</i> (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.</p

The effect of VOR on Fun-1 staining patterns of <i>C. tropicalis</i> cells within microcolonies.

<p><b>A</b>, <i>C. tropicalis</i> 2526 (VOR sensitive, MIC 0.0625 µg ml⁻¹) grown for 7 h on PAO (no-drug control). <b>B</b>, Microcolonies of the same strain cultured on 0.0625 µg VOR ml⁻¹ with an increase in Fun-1 staining (particularly for cells on the periphery). <b>C</b>, As panel <b>B</b>, showing the enhancement of Fun-1 staining of peripheral cells by 0.125 µg VOR ml⁻¹. Pictures were taken with identical exposure times (45 ms). Scale bar indicates 20 µm for all images.</p

Effect of staining procedure on calculation of microcolony area and MIC determination using <i>C. albicans</i> 4208.

1<p>area in µm².</p>2<p>calculated from log₁₀ microcolony area.</p>3<p>MIC in µg ml⁻¹.</p

Summary of susceptibility testing comparing PAO method with EUCAST and E-test methods.

<p>Results of testing the panel of 34 strains noted in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033818#s3" target="_blank">Methods</a> against AMB, CAS, ITR, VOR, and ANI. The percentage of exact matches was scored (S [sensitive] vs S, R [resistant] vs R, and where relevant I [intermediate] vs I) for pairwise comparisons between different tests and perfect agreement between all three tests.</p