A cell-free strategy for profiling of intracellular antibiotic sensitivity and resistance

Abstract

Antimicrobial resistance (AMR) is a pandemic spread across multiple priority infectious disease threats. Herein, we present a Klebsiella pneumoniae cell-free gene expression (CFE) system for the rapid profiling of intracellular antibiotic sensitivity and resistance. Specifically, this cell-free approach provides multiple antibiotic targets, which we exploit to identify which antibiotics are potent inside the cell but have limited transport. We also derive CFE systems from a selection of laboratory-adapted K. pneumoniae AMR variants, and as an exemplary case, show that a single RNA polymerase beta subunit variant H526L confers a 58-fold increase in CFE resistance to rifampicin. Overall, our cell-free model provides a safe (i.e., non-living) and rapid platform suitable for studying AMR variants in a Containment Level 1 laboratory. The approach is generalisable to laboratory and clinical strains and provides a new experimental tool that will significantly advance the understanding of antibiotic transport, AMR and provides a new infectious disease model.