Easy-to-use imaging-cytometry assay to analyze chitin patterns in yeasts.

• We developed a new assay for chitin content analysis in yeasts. • Our assay classified 12 yeast species based on chitin patterns and morphology. • These species were classified into the phylogenetic clades identified by Tsui. • The imaging-cytometry assay exhibited 85 % accuracy. Pathogenic fungi are a major threat to public health, and fungal infections are becoming increasingly common and treatment resistant. Chitin, a component of the fungal cell wall, modifies host immunity and contributes to antifungal resistance. Moreover, chitin content is regulated by chitin synthases and chitinases. However, the specific roles and mechanisms remain unclear. In this study, we developed a cytometric imaging assay to quantify chitin content and identify the distribution of chitin in the yeast cell wall. The Candida albicans SC5314 and Nakaseomyces glabratus (ex. C. glabrata) ATCC2001 reference strains, as well as 106 clinical isolates, were used. Chitin content, distribution, and morphological parameters were analysed in 12 yeast species. Moreover, machine learning statistical software was used to evaluate the ability of the cytometric imaging assay to predict yeast species using the values obtained for these parameters. Our imaging-cytometry assay was repeatable, reproducible, and sensitive to variations in chitin content in C. albicans mutants or after antifungal stimulation. The evaluated parameters classified the yeast species into the correct clade with an accuracy of 85 %. Our findings demonstrate that this easy-to-use assay is an effective tool for the exploration of chitin content in yeast species. [ABSTRACT FROM AUTHOR]