A technical pipeline for screening microbial communities as a function of substrate specificity through single cell fluorescent imaging

Abstract

The study of specific glycan uptake and metabolism has been shown to be an effective tool in aiding with the continued unravelling of the complexities in the human gut microbiome. To this aim fluorescent labelling of glycans may provide a powerful route towards target. In this study, we successfully used the fluorescent label 2-aminobenzamide (2-AB), most commonly employed for enhancing the detection of protein anchored glycans, to monitor and study microbial degradation of labelled glycans. Both single strain and co-cultured fermentations of microbes from the common human-gut derived Bacteroides genus, were able to grow when supplemented with 2-AB labelled glycans of different monosaccharide composition, degrees of acetylation and polymerization. Utilizing a multifaceted approach that combines chromatography, mass spectrometry, microscopy and flow cytometry techniques, it was possible to comprehensively track the metabolism of the labelled glycans in both supernatants and at a single cell level. We envisage this combination of complimentary techniques will help further the understanding of substrate specificity and the role it plays within microbial communities.