A low-footprint, fluorescence-based bacterial time-kill assay for estimating dose-dependent cell death dynamics.

Dose-response curves that describe the relationship between antibiotic dose and growth rate in bacteria are commonly measured with optical density (OD) based assays. While being simple and high-throughput, any dose-dependent cell death dynamics are obscured, as OD assays in batch culture can only quantify a positive net change in cells. Time-kill experiments can be used to quantify cell death rates, but current techniques are extremely resource-intensive and may be biased by residual drug carried over into the quantification assay. Here, we report a novel, fluorescence-based time-kill assay leveraging resazurin as a viable cell count indicator. Our method improves upon previous techniques by greatly reducing the material cost and being robust to residual drug carry-over. We demonstrate our technique by quantifying a dose-response curve in subject to cefotaxime, revealing dose-dependent death rates. We also show that our method is robust to extracellular debris and cell aggregation. Dose-response curves quantified with our method may provide a more accurate description of pathogen response to therapy, paving the way for more accurate integrated pharmacodynamic-pharmacokinetic studies.