Development and Optimization of a Higher-Throughput Bacterial Compound Accumulation Assay

The Gram-negative bacterial permeability barrier, coupled with efflux, raises formidable challenges to antibiotic drug discovery. The absence of efficient assays to determine compound penetration into the cell and impact of efflux makes the process resource-intensive, small-scale, and lacking much success. Here, we present BacPK: a label-free, solid phase extraction–mass spectrometry (SPE–MS)-based assay that measures total cellular compound accumulation in Escherichia coli. The BacPK assay is a 96-well accumulation assay that takes advantage of 9 s/sample SPE–MS throughput. This enables the analysis of each compound in a four-point dose–response in isogenic strain pairs along with a no-cell control and 16-point external standard curve, all in triplicate. To validate the assay, differences in accumulation were examined for tetracycline (Tet) and two analogs, confirming that close analogs can differ greatly in accumulation. Tet cellular accumulation was also compared for isogenic strains exhibiting Tet res...