Background: Culture-based MFC determination is cumbersome and time consuming. We developed a broth method of MFC determination based on drug removal, addition of fresh medium and assessment of growth of viable conidia adhered to the bottom of the well with XTT colorimetric assay of metabolic activity. Methods: After MIC determination of AMB and VRC for 16 A. fumigatus, 11 A. flavus and 13 A. terreus isolates by CLSI-M38A method in 3 replicate 96-well plates, MFC was assessed. From all clear wells of the 1st plate, 100 μl were subcultured on Sabouraud dextrose agar (SDA) plates and CFUs were counted after incubation for 48h (agar method). From all clear wells of the 2nd and 3rd plate the drug-containing supernatant was aspirated, the wells were washed with 200 μl saline and 200 μl RPMI was added. After 24 h incubation, 100 μg/ml XTT and 25 μM menadione were added, incubated for 2 h and absorbance measured at 450/630nm (broth method). The MFC was defined as the lowest of two consecutive drug concentrations yielding 0 CFU on SDA plates (agar method) or showing absorbance 8 mg/l (0.25->8) respectively for A. fumigatus, 2 mg/l (1->16) and 2 mg/l (0.5->8) for A. flavus and >16 mg/l (>16) and >8 mg/l (4->8) for A. terreus by agar method. The reproducibility of broth method was 92-97% and agreement (Â± 1 dilution) with agar method was 83-100% for both drugs and all species except for VRC and A. flavus (45-55%) where the agar method yielded lower MFCs than the broth method. However, when the remaining 100 μl from wells (1st plate) corresponding to VRC concentrations ≥ agar-defined MFC for A. flavus were aspirated and RPMI added, growth was detected at concentrations up to broth-defined MFCs suggesting overestimation of fungicidal activity with the agar method. Conclusions: The broth method can rapidly and reproducibly determine AMB and VRC MFC against Aspergillus spp.
Full conference title:
46th Interscience Conference on Antimicrobial Agents and Chemotherapy
- ICAAC 46th