An automated microscopy workflow to studyShigella–neutrophil interactions and antibiotic efficacyin 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 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 antibiotics reduce bacterial burden and not neutrophil recruitment to the hindbrain ventricle. We discover that nalidixic acid (a bactericidal antibiotic) can work with leukocytes in an additive manner to control Shigella flexneri infection, and also restrict dissemination of Shigella sonnei 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.

High-content high-resolution microscopy and deep learning assisted analysis reveals host and bacterial heterogeneity duringShigellainfection

“…Here, to dissect the complexity of S. flexneri infection in epithelial cells, we use high-content high-resolution microscopy coupled to automated image analysis. High-content microscopy enables the automated acquisition of thousands of microscopy images and therefore is useful to capture the heterogeneity of individual host and bacterial cells during the infection process for quantitative analysis (Aylan et al, 2023; Brodin & Christophe, 2011; Deboosere et al, 2021; Dramé et al, 2023; Fisch et al, 2019a; Lensen et al, 2023; Pylkkö et al, 2021). We analyse multiple parameters, such as morphological host cell features, the de novo synthesis of DNA and proteins in both host and bacterial cells, as well as activation of the bacterial T3SS.…”