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

Area of Science:

• Microbiology
• Immunology
• Neonatal Medicine

Background:

• The intestinal microbiota is crucial for host health, influencing digestion and immune system development.
• Current technologies primarily measure relative microbial abundance, limiting a deeper understanding of microbiome dynamics.
• Quantitative, single-cell analysis is needed to elucidate microbiome biology and compositional shifts.

Purpose of the Study:

• To introduce microbiota flow cytometry (MicFLY), a novel single-cell technology for precise bacterial quantification.
• To demonstrate MicFLY's capability in measuring absolute bacterial abundance at the species level.
• To investigate the association between specific bacterial populations and necrotizing enterocolitis (NEC) in preterm infants.

Main Methods:

• Development and application of microbiota flow cytometry (MicFLY) for single-cell bacterial analysis.
• Quantification of absolute bacterial abundance, differentiation of live/dead bacteria, and measurement of mRNA expression.
• Concurrent assessment of immunoglobulin A (IgA) and IgG binding to intestinal bacteria.

Main Results:

• MicFLY successfully quantified major intestinal taxa and assessed bacterial viability and gene expression.
• Longitudinal analysis in preterm infants revealed that increased IgA- and IgG-unbound Enterobacteriaceae species correlate with NEC development.
• The study identified specific bacterial changes associated with NEC pathogenesis.

Conclusions:

• MicFLY provides a powerful tool for high-resolution, quantitative microbiota measurements.
• This technology enables a deeper mechanistic understanding of microbiome shifts and their impact on host health.
• Findings highlight the potential of targeting unbound bacteria in NEC prevention or treatment strategies.