Background: The study focused on new tools to predict the mode of action of antifungals against Aspergillus fumigatus. The working hypothesis was that organelle-specific fluorescent microscopy and image analyses could be combined with NCCLS M-38P protocol to distinguish fungistatic and fungicidal effects. Methods: Amphotericin B (amp B), itraconazole (itra), and voriconazole (vori) MIC for 55 A. fumigatus clinical isolates were tested by NCCLS-M38P. MFC was determined from CFU counts on Sabouraud agar. Two A. fumigatus isolates with low-high MICs were further studied in 24-well plates with cover slips for microscopy and viability determination in fresh media post-treatment. The fluorescent probes were: Calcoflour white (cell wall), Propidium iodide (nuclei) and Mitotracker Green FM (mitochondria). The microscopy was with Nikon Optiphot with a Quad Fluor epifluorescence attachment. The optical sections were captured by aphotometrics PXL 1400 cooled CCD camera, processed and then stored on a SGI workstation using ISEE software. Results: The geometric mean MIC/MFC were lowest for vori (0.41/1.69 mg/ml), intermediate for amp B (2.0/3.60 mg/ml), and highest for itra (0.43/7.17 mg/ml). The control hypha showed discrete blue fluorescent walls and septa, red fluorescent nuclei, and many green fluorescent mitochondria. The maximal antifungal effects were visible 24 hrs post-treatment. Amp B MIC treated cells had shrunken hypha with organelle disintegrations and no viability. Vori MIC treated cells had hyphal swellings, organelle disintegration, and viability loss. Itra MIC treated cells showed swollen hypha, nuclear distortion, mitochondrial distintegration, and tested viable. Conclusions: Antifungal-treated A. fumigatus exhibited fragmentation of major organelles and hyphal disintegration, which correlated with the loss of viability for amp B and vori, while itra treated cells still retained viability.
Full conference title:
42nd Interscience Conference on Antimicrobial Agents and Chemotherapy
- ICAAC 42nd