High-throughput single-cell phenotypic profiling and backtracing exposes and predicts clinically relevant subpopulations in isogenic Staphylococcus aureus communities.

Isogenic bacterial cell populations are phenotypically heterogenous and may include subpopulations of antibiotic tolerant or heteroresistant cells. The reversible nature of these phenotypes and lack of biomarkers to differentiate functionally different, but morphologically identical cells is a challenge for research and clinical detection. To overcome this, we present Cellular Phenotypic Profiling and backTracing (CPPT), a flexible fluorescence-activated cell sorting platform, that uses optical probes to visualize and quantify cellular traits and connects the resulting phenotypic profile with a cells experimentally determined fate in single cell-derived growth and antibiotic susceptibility analysis. By applying CPPT on Staphylococcus aureus populations we recorded phenotypic signatures for dormant cells, exposed microanatomy-independent bimodal growth patterns in colony-derived cells, and revealed different culturability of single cells on solid compared to liquid media. We demonstrate that vancomycin-bodipyFL marks cellular subpopulations with increased likelihood to survive antibiotic exposure, showcasing the value of CPPT for discovery of clinically relevant biomarkers.