Single-cell quantification of the microbiota by flow cytometry: MicFLY

The intestinal microbiota regulates multiple host functions, including digestion and immune development. Our knowledge of the microbiota has been shaped by available technology that primarily measures relative abundance. However, understanding the basis of shifts in microbiota composition requires single cell, absolute abundance measurements. In response to this problem, we developed Microbiota Flow Cytometry (MicFLY), a single cell technology that directly quantifies and characterizes total bacterial abundances with species-level resolution in the microbiota. Using MicFLY, we can identify all major intestinal taxa, discriminate live from dead bacteria, perform single cell measurements of heterogeneous bacterial mRNA expression and concurrently quantify Immunoglobulin (Ig) A and G binding to intestinal bacteria. Using longitudinal species-resolved, quantitative analysis of the preterm infant microbiota, we identify that E. coli unbound by IgG and IgA associates with the development of necrotizing enterocolitis. The application of MicFLY single cell technology permits measurement of the microbiota at a finer scale and with deeper mechanistic understanding of compositional changes.

• Publication year

  2025

• Venue

  bioRxiv

• Publication date

  2025-09-21

• Fields of study

  Biology, Medicine, Environmental Science

• Identifiers
  DOI 10.1101/2025.09.18.677130PMID 41000908PMCID 12458334
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Highly multiplexed spatial transcriptomics in bacteria.

  2025cited by this paper
• Metagenomic immunoglobulin sequencing reveals IgA coating of microbial strains in the healthy human gut

  2025cited by this paper
• Propidium Monoazide is Unreliable for Quantitative Live–Dead Molecular Assays

  2025cited by this paper
• Absolute quantification of the living skin microbiome overcomes relic-DNA bias and reveals specific patterns across volunteers

  2025cited by this paper
• Clinical sequelae of gut microbiome development and disruption in hospitalized preterm infants.

  2024cited by this paper
• High-throughput single-microbe RNA sequencing reveals adaptive state heterogeneity and host-phage activity associations in human gut microbiome

  2024cited by this paper
• TEMPTED: time-informed dimensionality reduction for longitudinal microbiome studies

  2024cited by this paper
• Bacterial growth dynamics in a rhythmic symbiosis

  2024cited by this paper
• Single-cell transcriptomics across 2,534 microbial species reveals functional heterogeneity in the rumen microbiome

  2024cited by this paper
• Choice of DNA extraction method affects stool microbiome recovery and subsequent phenotypic association analyses

  2024cited by this paper
• Fecal microbial load is a major determinant of gut microbiome variation and a confounder for disease associations

  2024cited by this paper
• Highly accurate and sensitive absolute quantification of bacterial strains in human fecal samples

  2024cited by this paper
• Stability and heterogeneity in the antimicrobiota reactivity of human milk-derived immunoglobulin A

  2023cited by this paper
• MicrobiomeAnalyst 2.0: comprehensive statistical, functional and integrative analysis of microbiome data

  2023cited by this paper
• Quantifying bias introduced by sample collection in relative and absolute microbiome measurements

  2023cited by this paper
• Arresting microbiome development limits immune system maturation and resistance to infection in mice.

  2023cited by this paper
• Single-cell massively-parallel multiplexed microbial sequencing (M3-seq) identifies rare bacterial populations and profiles phage infection

  2023cited by this paper
• Murine Models of Salmonella Infection

  2023cited by this paper
• Probe-based bacterial single-cell RNA sequencing predicts toxin regulation

  2023cited by this paper
• Bacterial droplet-based single-cell RNA-seq reveals antibiotic-associated heterogeneous cellular states.

  2023cited by this paper
• Maternal gut microbiome–induced IgG regulates neonatal gut microbiome and immunity

  2022cited by this paper
• synDNA—a Synthetic DNA Spike-in Method for Absolute Quantification of Shotgun Metagenomic Sequencing

  2022cited by this paper
• Statistical challenges in longitudinal microbiome data analysis

  2022cited by this paper
• Neutrophils discriminate live from dead bacteria by integrating signals initiated by Fprs and TLRs

  2022cited by this paper
• Spatial transcriptomics of planktonic and sessile bacterial populations at single-cell resolution

  2021cited by this paper
• Unigems: plasmids and parts to facilitate teaching on assembly, gene expression control and logic in E. coli

  2021cited by this paper
• Segment number threshold determines juvenile onset of germline cluster expansion in Platynereis dumerilii

  2021cited by this paper
• Multi-kingdom ecological drivers of microbiota assembly in preterm infants

  2021cited by this paper
• Primer, Pipelines, Parameters: Issues in 16S rRNA Gene Sequencing

  2021cited by this paper
• Temporal variability in quantitative human gut microbiome profiles and implications for clinical research

  2021cited by this paper
• Whole microbial community viability is not quantitatively reflected by propidium monoazide sequencing approach

  2020cited by this paper
• Context-aware dimensionality reduction deconvolutes gut microbial community dynamics

  2020cited by this paper
• Single-cell RNA-sequencing reports growth-condition-specific global transcriptomes of individual bacteria

  2020cited by this paper
• Assessment of fecal DNA extraction protocols for metagenomic studies

  2020cited by this paper
• Multiplexed Quantitative In Situ Hybridization for Mammalian or Bacterial Cells in Suspension: qHCR Flow Cytometry (v3.0).

  2020cited by this paper
• Influence of Maternal Milk on the Neonatal Intestinal Microbiome

  2020cited by this paper
• The Impact of Human Milk on Necrotizing Enterocolitis: A Systematic Review and Meta-Analysis

  2020cited by this paper
• Quantitative PCR provides a simple and accessible method for quantitative microbiota profiling

  2020cited by this paper
• Trends in incidence and outcomes of necrotizing enterocolitis over the last 12 years: A multicenter cohort analysis.

  2020cited by this paper
• Single-Cell Analysis Reveals that the Enterococcal Sex Pheromone Response Results in Expression of Full-Length Conjugation Operon Transcripts in All Induced Cells

  2020cited by this paper
• Comparison of bias and resolvability in single-cell and single-transcript methods

  2020cited by this paper
• The gut bacterium Extibacter muris produces secondary bile acids and influences liver physiology in gnotobiotic mice

  2020cited by this paper
• Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis

  2019cited by this paper
• Maternal IgA protects against the development of necrotizing enterocolitis in preterm infants

  2019cited by this paper
• The Impact of DNA Extraction Methods on Stool Bacterial and Fungal Microbiota Community Recovery

  2019cited by this paper
• Prokaryotic Single-Cell RNA Sequencing by In Situ Combinatorial Indexing

  2019cited by this paper
• Microbial single-cell RNA sequencing by split-pool barcoding

  2019cited by this paper
• Quantitative Visualization of Gene Expression in Mucoid and Nonmucoid Pseudomonas aeruginosa Aggregates Reveals Localized Peak Expression of Alginate in the Hypoxic Zone

  2019cited by this paper
• Using fluorescence flow cytometry data for single-cell gene expression analysis in bacteria

  2019cited by this paper
• Effect of Holder pasteurization on macronutrients and immunoglobulin profile of pooled donor human milk

  2019cited by this paper
• Dead or alive: how the immune system detects microbial viability.

  2019cited by this paper
• Necrotizing enterocolitis is preceded by increased gut bacterial replication, Klebsiella, and fimbriae-encoding bacteria

  2019cited by this paper
• Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust

  2018cited by this paper
• Intestinal microbiota development and gestational age in preterm neonates

  2018cited by this paper
• Sensing Microbial Viability through Bacterial RNA Augments T Follicular Helper Cell and Antibody Responses

  2018cited by this paper
• Absolute quantitation of microbiota abundance in environmental samples

  2018cited by this paper
• Quantification of variation and the impact of biomass in targeted 16S rRNA gene sequencing studies

  2018cited by this paper
• Subcellular antigen localization in commensal E. coli is critical for T cell activation and induction of specific tolerance

  2018cited by this paper
• Controlling for Contaminants in Low-Biomass 16S rRNA Gene Sequencing Experiments

  2018cited by this paper
• Multiplexed in situ protein imaging using DNA-barcoded antibodies with extended hybridization chain reactions

  2018cited by this paper
• STING Senses Microbial Viability to Orchestrate Stress-Mediated Autophagy of the Endoplasmic Reticulum.

  2017cited by this paper
• Bacteria and Bioactivity in Holder Pasteurized and Shelf-Stable Human Milk Products

  2017cited by this paper
• Towards standards for human fecal sample processing in metagenomic studies

  2017cited by this paper
• Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water

  2017cited by this paper
• Assessment of variation in microbial community amplicon sequencing by the Microbiome Quality Control (MBQC) project consortium

  2017cited by this paper
• Genomic GC-Content Affects the Accuracy of 16S rRNA Gene Sequencing Based Microbial Profiling due to PCR Bias

  2017cited by this paper
• Stochasticity in the enterococcal sex pheromone response revealed by quantitative analysis of transcription in single cells

  2017cited by this paper
• Intestinal dysbiosis in preterm infants preceding necrotizing enterocolitis: a systematic review and meta-analysis

  2017cited by this paper
• Quantitative microbiome profiling links gut community variation to microbial load

  2017cited by this paper
• Microbiome Datasets Are Compositional: And This Is Not Optional

  2017cited by this paper
• Microbiome-wide association studies link dynamic microbial consortia to disease

  2016cited by this paper
• Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples.

  2016cited by this paper
• Development of the gut microbiota and mucosal IgA responses in twins and gnotobiotic mice

  2016cited by this paper
• Developmental dynamics of the preterm infant gut microbiota and antibiotic resistome

  2016cited by this paper
• Temporal bacterial and metabolic development of the preterm gut reveals specific signatures in health and disease

  2016cited by this paper
• Systematic improvement of amplicon marker gene methods for increased accuracy in microbiome studies

  2016cited by this paper
• Compositional data analysis of the microbiome: fundamentals, tools, and challenges.

  2016cited by this paper
• Toward Accurate and Quantitative Comparative Metagenomics.

  2016cited by this paper
• The Effect of Holder Pasteurization on Nutrients and Biologically-Active Components in Donor Human Milk: A Review

  2016cited by this paper
• Metagenomic Sequencing with Strain-Level Resolution Implicates Uropathogenic E. coli in Necrotizing Enterocolitis and Mortality in Preterm Infants

  2016cited by this paper
• Absolute quantification of microbial taxon abundances

  2016cited by this paper
• Mapping a multiplexed zoo of mRNA expression

  2016cited by this paper
• Development of a robust immuno‐S‐FISH protocol using imaging flow cytometry

  2016cited by this paper
• Adjusting microbiome profiles for differences in microbial load by spike-in bacteria

  2016cited by this paper
• Stool bacterial load in preterm infants with necrotising enterocolitis.

  2016cited by this paper
• Gut Microbiota-Induced Immunoglobulin G Controls Systemic Infection by Symbiotic Bacteria and Pathogens.

  2016cited by this paper
• Microbiota Diurnal Rhythmicity Programs Host Transcriptome Oscillations.

  2016cited by this paper
• Maternal IgG and IgA Antibodies Dampen Mucosal T Helper Cell Responses in Early Life.

  2016cited by this paper
• The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies

  2015cited by this paper
• Use of Hybridization Chain Reaction-Fluorescent In Situ Hybridization To Track Gene Expression by Both Partners during Initiation of Symbiosis

  2015cited by this paper
• Rhythmicity of the intestinal microbiota is regulated by gender and the host circadian clock

  2015cited by this paper
• Gut bacteria are rarely shared by co-hospitalized premature infants, regardless of necrotizing enterocolitis development

  2015cited by this paper
• Functional characterization of IgA-targeted bacterial taxa from undernourished Malawian children that produce diet-dependent enteropathy

  2015cited by this paper
• Patterned progression of bacterial populations in the premature infant gut

  2014cited by this paper
• Bacterial sugar utilization gives rise to distinct single‐cell behaviours

  2014cited by this paper
• The treatment-naive microbiome in new-onset Crohn's disease.

  2014cited by this paper
• Next-Generation in Situ Hybridization Chain Reaction: Higher Gain, Lower Cost, Greater Durability

  2014cited by this paper
• Transkingdom control of microbiota diurnal oscillations promotes metabolic homeostasis.

  2014cited by this paper
• Noncanonical inflammasome activation of caspase-4/caspase-11 mediates epithelial defenses against enteric bacterial pathogens.

  2014cited by this paper
• The Impact of Different DNA Extraction Kits and Laboratories upon the Assessment of Human Gut Microbiota Composition by 16S rRNA Gene Sequencing

  2014cited by this paper

• No citing papers are available for this paper.