Single-cell detection and quantification of the microbiota by MicFLY.

The intestinal microbiota regulates multiple host functions, including digestion and immune development. Our knowledge of the microbiota has been shaped by available technology, which primarily measures relative abundance. Quantitative, single-cell bacterial measurements would improve our understanding of microbiome biology, including the dynamics behind shifts in microbiota composition. We present microbiota flow cytometry (MicFLY), a single-cell technology that quantifies absolute bacterial abundance with species-level resolution. Using MicFLY, we can identify the major intestinal taxa, discriminate live from dead bacteria, measure heterogeneous mRNA expression, and concurrently quantify immunoglobulin A (IgA) and IgG binding to intestinal bacteria. Using longitudinal, quantitative microbiota analyses on a small cohort of preterm infants, we find that increases in IgA- and IgG-unbound bacteria from various Enterobacteriaceae species associate with the development of necrotizing enterocolitis (NEC). MicFLY single-cell technology thus enables fine-scale quantitative microbiota measurements for a deeper mechanistic understanding of compositional changes.