Use of Fluorescent Probes To Determine MICs of Amphotericin B and Caspofungin against Candida spp. and Aspergillus spp

Abstract

We investigated the utility of mechanism-based fluorescent probes for determination of MICs (FMICs) of amphotericin B and caspofungin against Candida spp. and Aspergillus spp. Amphotericin B was selected as a membrane-active antifungal agent, and caspofungin was selected as a cell wall-active agent. FMICs were also compared to the MIC determined by CLSI (formerly NCCLS) methods. Five isolates per species of Candida albicans, Candida glabrata, Candida parapsilosis, Aspergillus fumigatus, and Aspergillus terreus were studied with either amphotericin B or caspofungin. The fluorescent probes, carboxyfluorescein diacetate (CFDA) for cytoplasmic esterase activity and dihexyloxacarbocyanine iodide (DiOC 6 ) for cell membrane potential, were each added to their respective plates. MICs and FMICs were determined in at least three separate experiments (in duplicate). Fluorescence was measured using a 96-well plate fluorometer. For amphotericin B and caspofungin, the FMIC end point was the lowest concentration of drug at which the percent growth inhibition from treated organisms versus control organisms displayed 80% inhibition for amphotericin B and 50% inhibition for caspofungin as measured by a fluorescent signal. The MIC for amphotericin B was defined as the lowest concentration of antifungal displaying no visible growth for both Aspergillus and Candida spp. The MIC for caspofungin was the lowest concentration of drug that displayed a minimum effective concentration for Aspergillus spp. For Candida spp., the MIC for caspofungin was defined as the concentration at which the antifungal agent significantly inhibits the organism. The FMICs of both antifungals, as measured by the DiOC 6 membrane probe, showed good agreement (83% to 100%), within one well dilution, with the MICs against amphotericin B and caspofungin for all species. Also, the FMICs measured by the CFDA cytoplasmic esterase probe reflecting damage due to cell wall or cell membrane showed strong agreement (79 to 100%) with the MICs of both amphotericin B and caspofungin for all species. There was no significant difference in comparisons of MIC and FMIC values (P > 0.05). The use of fluorescent probes provides a mechanism-based method of determination of MICs of amphotericin B and caspofungin against Candida spp. and Aspergillus spp. that correlates well with standard methods.Recent advances in fluorescent-probe technology may permit the use of these reagents in assessing the alteration of physiologic function due to antifungal agents (1,5,6,11,12,23). Therefore, we investigated the utility of a fluorometric assay using the fluorescent probes carboxyfluorescein diacetate (CFDA) and dihexyloxacarbocyanine iodide (DiOC 6 ) to assess the in vitro antifungal activities of amphotericin B deoxycholate representing the class of membrane-active polyenes and caspofungin representing the class of cell wall-active echinocandins (7).The CFDA probe is taken up by viable cells and upon hydrolysis by intracellular esterases, forms carboxyfluorescein. The carboxyfluorescein is...

Screening method to identify inhibitors of siderophore biosynthesis in the opportunistic fungal pathogen,Aspergillus fumigatus

“…Because no specific inhibitor of siderophore biosynthesis has yet been identified, the practical application of this screening method was demonstrated using a known antifungal agent (amphotericin B) (Table 1). The MIC for amphotericin B (0·5 μ g ml −1 ) was the same as the value obtained by several investigators using standard NCCLS methodology (Peter et al. 2005) and validates our conditions for determining active antifungal agents.…”

“…Because no specific inhibitor of siderophore biosynthesis has yet been identified, the practical application of this screening method was demonstrated using a known antifungal agent (amphotericin B) ( Table 1). The MIC for amphotericin B (0AE5 lg ml )1 ) was the same as the value obtained by several investigators using standard NCCLS methodology (Peter et al 2005) and validates our conditions for determining active antifungal agents. As expected, no effect of iron on growth inhibition by amphotericin B was observed (Table 1) because the mechanism of inhibition by amphotericin B is unrelated Table 1 Results from Level 1 and Level 2 screens using a known antifungal agent (amphotericin B)…”

Fluorescent Tracking of Yeast Division Clarifies the Essential Role of Spleen Tyrosine Kinase in the Intracellular Control of Candida glabrata in Macrophages

“…Esterases are necessary for the activation of CFSE for covalent conjugation to amino groups on proteins. In mammalian cells, esterases are abundant within the cytoplasm, whereas in yeast, high levels of esterases are found within the fungal cell wall ( 34 ). This esterase distribution clarifies the reason for concentrated CFSE conjugation to the cell wall ultrastructure as opposed to homogenous cytoplasmic staining as seen in lymphocytes.…”