High-throughput time-resolved morphology screening in bacteria reveals phenotypic responses to antibiotics.

Image-based high-throughput screening strategies for quantifying morphological phenotypes have proven widely successful. Here we describe a combined experimental and multivariate image analysis approach for systematic large-scale phenotyping of morphological dynamics in bacteria. Using off-the-shelf components and software, we established a workflow for high-throughput time-resolved microscopy. We then screened the single-gene deletion collection of Escherichia coli for antibiotic-induced morphological changes. Using single-cell quantitative descriptors and supervised classification methods, we measured how different cell morphologies developed over time for all strains in response to the β-lactam antibiotic cefsulodin. 191 strains exhibit significant variations under antibiotic treatment. Phenotypic clustering provided insights into processes that alter the antibiotic response. Mutants with stable bulges show delayed lysis, contributing to antibiotic tolerance. Lipopolysaccharides play a crucial role in bulge stability. This study demonstrates how multiparametric phenotyping by high-throughput time-resolved imaging and computer-aided cell classification can be used for comprehensively studying dynamic morphological transitions in bacteria.