Large-Scale Expansion of the MALDI-TOF MS Library for Comprehensive Identification of Yeasts and Filamentous Fungi in...

The rapid identification of microbes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is gaining attention, particularly in clinical laboratories and hygiene management in food manufacturing. However, unlike bacteria, technical issues related to preprocessing and a lack of comprehensive reference libraries pose challenges in fungi. In this study, we constructed a new MALDI-TOF MS database, named EMALiMB, that expands the existing reference library to accurately identify a wider range of microbial species. The new reference library included 75 genera and 430 species of Ascomycota, 77 genera and 213 species of Basidiomycota, i.e. a total of 643 species of fungi, and six species belonging to the genus Prototheca. All strains were selected to complement taxa that were either not registered in the current library or were insufficiently represented, owing to a small number of strains. For example, 107 Candida species included pathogens, but also non-pathogenic species, inhabiting in the environment, and phylogenetically closely related with clinical relevants. Additionally, we improved the ionization of basidiomycetous yeasts and filamentous species, and Trichophyton, which had not been sufficiently ionized before, by incorporating bead-crushing in the pretreatment. The accuracy of this new reference library was evaluated using 384 clinical and environmental yeast isolates. A slight but remarkable increase in accuracy from 85.20% to 87.28% and in the mean score from 2.15 to 2.27 was obtained. The coverage rate for tested species improved significantly, from 80% to 88.57% for clinically relevant species, and from 52.38% to 76.19% for species isolated from environments.