Author:

Keywords:

candida-albicans, testing susceptibilities, in-vitro, caspofungin, fluconazole, voriconazole, assay, identification, surveillance, itraconazole, Science & Technology, Life Sciences & Biomedicine, Microbiology, Pharmacology & Pharmacy, CANDIDA-ALBICANS, TESTING SUSCEPTIBILITIES, IN-VITRO, CASPOFUNGIN, FLUCONAZOLE, VORICONAZOLE, ASSAY, IDENTIFICATION, SURVEILLANCE, ITRACONAZOLE, Amphotericin B, Antifungal Agents, Candida, Candida albicans, Caspofungin, Echinocandins, Indicator Dilution Techniques, Kinetics, Lipopeptides, Microbial Sensitivity Tests, Nuclear Magnetic Resonance, Biomolecular, Peptides, Cyclic, Pyrimidines, Triazoles, Trichosporon, Voriconazole, 0605 Microbiology, 1108 Medical Microbiology, 1115 Pharmacology and Pharmaceutical Sciences, 3107 Microbiology, 3207 Medical microbiology, 3214 Pharmacology and pharmaceutical sciences

Abstract:

Drug-induced inhibition of fungal growth is used in the diagnostic laboratory to predict therapeutic efficacy but is relatively slow, and determination of endpoints can be problematic. Nuclear magnetic resonance (NMR) spectroscopy identifies the metabolic complement of microorganisms while monitoring utilization of constituents of the incubation medium. This technique may provide a rapid and objective indicator of antifungal effects. We evaluated the effects of caspofungin, amphotericin B (AMB), and voriconazole on metabolic profiles of yeast species cultured in RPMI-2% glucose-morpholinepropanesulfonic acid buffer in microtiter plates in a proof-of-principle study. H-1 NMR spectra were obtained using Bruker NMR spectrometers at H-1 frequencies of 600 and 360 MHz. Metabolites were identified by two-dimensional correlation NMR spectra, and relative peak integrals were calculated from one-dimensional H-1 NMR spectra. MICs were determined by a modification of the Clinical and Laboratory Standards Institute broth microdilution method M27-A. Utilization of glucose and branched-chain and aromatic amino acid substrates was accompanied by fungal production of acetate, acetaldehyde, ethanol, formate, fumarate, glycerol, lactate, pyruvate, and succinate. Clear-cut metabolic endpoints indicating a greater than 50% reduction in substrate utilization and fungal metabolite production which correlated with MICs were noted at 16 and 24 h for all three drugs. At 8 h, reductions of greater than 50% for selected metabolites were noted for caspofungin and AMB. Direct NMR-based observation of metabolic alterations in yeast cultures reveals changes in key metabolic pathways and should be evaluated formally as a rapid technique for determining susceptibility to antifungal drugs.