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Characterizing Microbiome Dynamics – Flow Cytometry Based Workflows from Pure Cultures to Natural Communities
Published on: July 12, 2018
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Single-cell quantification of the microbiota by flow cytometry: MicFLY.
Christine M Tin1,2,3, Bianca Cordazzo Vargas4, Darryl A Abbott1,2,3
1Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA.
Biorxiv : the Preprint Server for Biology
|September 26, 2025
Summary
A new technology, Microbiota Flow Cytometry (MicFLY), enables precise, single-cell measurement of gut bacteria. This innovation links specific bacterial interactions, like unbound E. coli, to infant necrotizing enterocolitis development.
Area of Science:
- Microbiology
- Immunology
- Bioengineering
Background:
- The intestinal microbiota is crucial for host health, influencing digestion and immune system development.
- Current technologies primarily measure relative bacterial abundance, limiting a deep understanding of microbiota shifts.
- Absolute abundance and single-cell measurements are needed to understand microbiota composition changes.
Purpose of the Study:
- To develop a novel single-cell technology for quantifying and characterizing gut bacteria with species-level resolution.
- To enable detailed analysis of bacterial absolute abundance, viability, gene expression, and host immune molecule binding.
- To investigate the relationship between specific bacterial features and necrotizing enterocolitis in preterm infants.
Main Methods:
- Development and application of Microbiota Flow Cytometry (MicFLY), a single-cell technology.
- Quantification of total bacterial abundances at the species level.
- Discrimination of live vs. dead bacteria and measurement of bacterial mRNA expression.
- Concurrent quantification of Immunoglobulin (Ig) A and G binding to intestinal bacteria.
Main Results:
- MicFLY successfully quantifies and characterizes total bacterial abundances with species-level resolution.
- The technology allows for live/dead discrimination, single-cell mRNA expression analysis, and Ig binding quantification.
- Longitudinal analysis revealed that E. coli unbound by IgG and IgA is associated with necrotizing enterocolitis in preterm infants.
Conclusions:
- MicFLY provides unprecedented single-cell resolution for quantitative microbiota analysis.
- This technology offers deeper mechanistic insights into microbiota compositional changes and host-microbe interactions.
- MicFLY application identified a specific bacterial-immune association linked to necrotizing enterocolitis, paving the way for improved diagnostics and therapeutics.
