An automated microscopy workflow to study Shigella-neutrophil interactions and antibiotic efficacy in vivo

ABSTRACT Shigella are Gram-negative bacterial pathogens responsible for bacillary dysentery (also called shigellosis). The absence of a licensed vaccine and widespread emergence of antibiotic resistance has recently led the WHO to highlight Shigella as a priority pathogen requiring urgent attention. Several infection models have been useful to explore the Shigella infection process, yet we still lack information regarding events taking place in vivo . Here, using a Shigella -zebrafish infection model and high-content microscopy, we develop an automated microscopy workflow to non-invasively study fluorescently labelled bacteria and neutrophils in vivo . We apply our workflow to antibiotic-treated zebrafish and demonstrate that neutrophil recruitment is independent of bacterial burden. Strikingly, we discover that nalidixic acid (a bactericidal antibiotic) can restrict Shigella dissemination from the hindbrain ventricle. We envision that our automated microscopy workflow, applied here to study Shigella -neutrophil interactions and antibiotic efficacy in zebrafish, can be useful to innovate treatments for infection control in humans. SUMMARY STATEMENT We develop an automated image analysis workflow to enable fast and reliant immune cell counting in Shigella -infected zebrafish larvae, and reveal how antibiotics impact Shigella -neutrophil interactions in vivo .