Abstract
Development of new and improved tuberculosis (TB) chemotherapies is hampered by antibiotic resistance and drug tolerance by Mycobacterium tuberculosis (Mtb). Phenotypic drug tolerance, a phenomenon where Mtb populations can temporarily survive therapeutic antibiotic concentrations, represents a significant hurdle to TB treatment and is indeed one of the factors responsible for prolonged TB therapy. Assays that can identify compounds with improved efficacy against drug-tolerant Mtb are urgently required to improve TB treatment regimens. Here, we report the development of a 96-well plate assay capable of identifying anti-Mtb drugs with activity against drug-tolerant Mtb in physiologically relevant intracellular environments within macrophages. Primary murine macrophages, modified either by immunological activation or specific CRISPR/Cas9 gene knockouts to generate tolerance-inducing environments, were infected with an Mtb strain constitutively expressing luciferase. Following drug exposure, differences in bacterial survival were measured by bacterial outgrowth after lysis of the host macrophages. By monitoring Mtb luciferase in infected macrophages before, during, and after drug treatment, we confirmed earlier observations that host immune stresses trigger induction of drug tolerance. However, while host stresses induced tolerance against some anti-TB compounds, the same host stresses were synergistic with other anti-TB drugs. Our assay provides the ability to profile the activities of anti-TB drugs on bacteria in intracellular host environments, which is critical to the rational design of drug combinations that provide optimal coverage of the Mtb sub-populations in the infected host.