Antimicrobial assay optimization and validation for HTS in 384-well format using a bioluminescent E. coli K-12 strain.

This report describes the optimization and validation of an antimicrobial assay based on the genetically modified bacterial strain Escherichia coli K-12 (pTetlux1). The use of this particular strain enables an inducible cell-based bioluminescent assay for high-throughput screening (HTS) of antimicrobial agents, which shows a pronounced detection of compounds targeting transcriptional and translational events in protein synthesis. The optimizations in 96-well format led to several improvements in assay conditions, such as reduction of the pre-incubation time before luminescence induction by half. The threshold for DMSO tolerability was concluded to be up to 1%. Assay protocol was further miniaturized into 384-well format and the liquid handling was automated using a robotic workstation. The use of compound pre-plating into 384-well plates as a part of the process was evaluated, and the total assay volume was further downscaled from 50 μl to 30 μl. With this approach, the amount of test compound needed per well was reduced to nanoliter volumes. Using the miniaturized protocol a pilot screen of 2000 known drugs and bioactives was performed. The assay performance was evaluated by calculating known assay quality parameters, the Z' factor having a mean value of 0.8 during the compound library screening indicated an excellent performance. Of the assay positives, 54 compounds showed high inhibitions (60-100%), of which the majority (89%) were known antibacterial agents. Of the actives showing >60% inhibition, 16 compounds were identified as known transcriptional and translational inhibitors. The screening results demonstrated that the miniaturized assay is well suited for identification of antimicrobial compounds in HT screening, and that the assay is specifically sensitive towards bacterial transcription and translation inhibitors.