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

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.