OPTIKA, a new high content kill-kinetic assay to longitudinally assess in vitro drug combinations against Mycobacterium tuberculosis

Abstract In vitro methods to characterize drug combinations typically involve phenotypic screenings using checkerboard assays (CBA) or, more recently, DiaMOND. Such approaches rely on the Fractional Inhibitory Concentration Index (FICI), a fixed-time measurement of growth inhibition that, nonetheless, necessitates secondary validation by time-kill assays (TKA). Longitudinal time-kinetics of bacterial killing are considered the gold standard in vitro proxy for antimicrobial activity, but they required increased assay complexity, particularly against the slow growing Mycobacterium tuberculosis. Here, we developed a new methodology named OPTIKA (Optimized Time Kill Assays) that enhances the capacity of traditional TKA by over 1000-fold. This allows for easy and dynamic examination of n-way drug interactions by simultaneously monitoring bactericidal and sterilizing capacities in a longitudinal manner. We then replicated previous DiaMOND studies and performed comparisons using CBA and OPTIKA methodologies. We demonstrate that selection of the efficacy parameters (either routed on bacteriostatic, bactericidal or sterilizing properties) affects the interpretation of in vitro drug interactions and, consequently, its potential translational value. The increased assay throughput provided by OPTIKA offers a novel framework for developing tuberculosis treatment regimens. Teaser OPTIKA is a new methodology that increases time-kill assay performance against Mycobacterium tuberculosis by over 1,000-fold Competing Interest Statement MPAC, PG, AML, SFG, and RGdR are, or were, employees of GSK, a pharmaceutical company working in the development of novel anti-TB drugs. AML and RGdR hold shares in GSK. SRG declare no conflicts of interest. All authors approved the submission of the document.