Work

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

Year: 2026

Type: article

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.

Source: MethodsX

DOI: 10.1016/j.mex.2026.104194

Authors: Hasna Toukabri, Christophe Buisson, Yves Le Vern, Alix Sausset, Mickaël Bourge, Leyla Slamti