Summary

In vitro antibiotic testing guides therapy and drug development, but conventional growth inhibition assays often fail to predict treatment responses. We developed Antimicrobial Single-Cell Testing (ASCT), a large-scale live-cell imaging approach that quantifies bacterial killing in real time at single-cell resolution. Tracking 140 million mycobacteria across 20,000 time-kill curves, we identified key determinants of antibiotic killing and its clinical relevance. In Mycobacterium tuberculosis, we show that drug-specific killing in starved bacteria, rather than growth inhibition or killing of growing cells, predicts regimen efficacy in mice and humans. Extending ASCT to 405 Mycobacterium abscessus strains showed that antibiotic killing (drug tolerance) is genetically encoded. We show that tolerance patterns cluster by antibiotic targets, identify a phage protein modulating antibiotic killing, and link strain-specific killing dynamics to individual patient outcomes independent of drug resistance. Together, our findings reveal how drug properties and bacterial diversity shape treatment responses, offering a path to more effective, personalised therapies.