Chemical pretreatment of cells for enhanced discrimination of clinical yeast isolates by MALDI-TOF-MS

Abstract

BACKGROUND: Candida spp. of yeasts are a major cause of nosocomial infections in intensive care units, with Candida albicans the most commonly isolated yeast species. Phenotypic identification of Candida spp. is labour- and time consuming, while rapid direct intact cell MALDI-TOF-MS is emerging as a rapid tool for identifying microorganisms.

OBJECTIVES: To optimise a MALDI-TOF-MS protocol for discrimination of yeasts, with evaluation of various chemical pretreatments of cells for enhancing spectral richness and discriminatory power.

METHOD: MALDI-TOF-MS of C. albicans was optimised with respect to matrix chemical(s), matrix solvent and target plating method. Various chemical pretreatments (solvents, reductants, detergents) and application methods were evaluated for enhancement of spectral richness. Selected pretreatments were applied to MALDI-TOF-MS discrimination of a set of clinical isolates comprising Candida spp. and Cryptococcus neoformans.

RESULTS: The optimized MALDI-TOF-MS protocol involved α-cyano-4-hydroxycinnamic acid (CHCA) as matrix chemical (in 1:1 acenonitrile:H₂O and 0.3% trifluoroacetic acid), with target plating by a sandwich method. Methanol or formate:isopropanol pretreatment of cells in a direct target plate application method enhanced spectral richness. The latter pretreatment facilitated discrimination of a set of clinical isolates comprising Candida spp. (C. albicans, C. boidinii, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, C. tropicalis) and Cryptococcus neoformans with 100% clustering accuracy.

CONCLUSION: MALDI-TOF-MS discrimination of Candida spp. is enhanced through chemical pretreatment of cells; formate:isopropanol pretreatment, with CHCA as matrix chemical in a sandwich target plating method, is highlighted as a protocol affording the best discriminatory power.