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Peer-reviewedCC BY 4.0MethodsX

FACS-based isolation and recovery of physiologically distinct bacterial subpopulations from infected insect hosts

  • Hasna Toukabri1
  • Christophe Buisson1
  • Yves Le Vern2
  • Alix Sausset2
  • Mickaël Bourge3
  • Leyla Slamti1
  1. 1Micalis Institute, INRAE, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, 78350, France.
  2. 2Unité Mixte de Recherches (UMR) 1282 Infectiologie et Santé Publique, INRAE Centre Val de Loire, Université François Rabelais de Tours, Nouzilly, France.
  3. 3Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France.
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Abstract

Phenotypic heterogeneity is often encountered during infection but remains challenging to investigate in vivo. Here, using the entomopathogenic spore-former Bacillus thuringiensis with the Galleria mellonella insect larva infection model and Fluorescence-Activated Cell Sorting (FACS), we present a workflow that enables the isolation of bacterial subpopulations in distinct physiological states, directly from infected hosts, for downstream characterization. Host-derived debris and the low bacterial recovery after sorting represent major limitations of in vivo-derived FACS approaches. By combining pre-sorting filtration, optimized FACS parameters, and post-sorting processing, the workflow minimizes host-derived contamination while preserving bacterial viability and physiological integrity. The recovered subpopulations are suitable for a wide range of downstream characterization, including reinfection experiments, tolerance assays, infection cycle studies and competition assays. As an illustrative application, we used this method on larvae infected with a strain harboring a fluorescent reporter that marks cells irreversibly committed to sporulation. Sporulating and non-sporulating bacteria recovered from the insects were successfully separated and subsequently used in reinfection experiments. • Isolation of physiologically distinct bacterial subpopulations directly from infected insect hosts • FACS-based separation of bacterial subpopulations using fluorescent reporters • Recovery of viable sorted bacterial populations for downstream characterization.

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Cite

BibTeXRIS
BibTeX
@article{toukabri2026facsbased,
  title = {{FACS-based isolation and recovery of physiologically distinct bacterial subpopulations from infected insect hosts}},
  author = {Hasna Toukabri and Christophe Buisson and Yves Le Vern and Alix Sausset and Mickaël Bourge and Leyla Slamti},
  journal = {MethodsX},
  year = {2026},
  month = oct,
  doi = {10.1016/j.mex.2026.104194},
  url = {https://hal.inrae.fr/hal-05839735v1},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - FACS-based isolation and recovery of physiologically distinct bacterial subpopulations from infected insect hosts
AU  - Hasna Toukabri
AU  - Christophe Buisson
AU  - Yves Le Vern
AU  - Alix Sausset
AU  - Mickaël Bourge
AU  - Leyla Slamti
PY  - 2026
DA  - 2026/10/07
JO  - MethodsX
DO  - 10.1016/j.mex.2026.104194
UR  - https://hal.inrae.fr/hal-05839735v1
AB  - Phenotypic heterogeneity is often encountered during infection but remains challenging to investigate in vivo. Here, using the entomopathogenic spore-former Bacillus thuringiensis with the Galleria mellonella insect larva infection model and Fluorescence-Activated Cell Sorting (FACS), we present a workflow that enables the isolation of bacterial subpopulations in distinct physiological states, directly from infected hosts, for downstream characterization. Host-derived debris and the low bacterial recovery after sorting represent major limitations of in vivo-derived FACS approaches. By combining pre-sorting filtration, optimized FACS parameters, and post-sorting processing, the workflow minimizes host-derived contamination while preserving bacterial viability and physiological integrity. The recovered subpopulations are suitable for a wide range of downstream characterization, including reinfection experiments, tolerance assays, infection cycle studies and competition assays. As an illustrative application, we used this method on larvae infected with a strain harboring a fluorescent reporter that marks cells irreversibly committed to sporulation. Sporulating and non-sporulating bacteria recovered from the insects were successfully separated and subsequently used in reinfection experiments. • Isolation of physiologically distinct bacterial subpopulations directly from infected insect hosts • FACS-based separation of bacterial subpopulations using fluorescent reporters • Recovery of viable sorted bacterial populations for downstream characterization.
SN  - 2215-0161
ER  -