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

Abstract

AbstractAntimicrobial resistance (AMR) is a pandemic spread across multiple priority infectious disease threats. While the cell envelope plays a key role in AMR, this also makes it challenging to study how antibiotics function inside the cell. Herein, we present aKlebsiella pneumoniaecell-free gene expression (CFE) platform for the rapid profiling of intracellular antibiotic sensitivity and resistance. This cell-free approach provides the unique macromolecular and metabolite components from this microbe, which include multiple antibiotic targets from transcription, translation, and metabolic processes. First, we compare theK. pneumoniaeCFE system to whole cell antimicrobial assays. We find that several antibiotic classes show higher sensitivity in the CFE system, suggesting limitations in antibiotic transport in the whole cell assay. Next, we evolvedK. pneumoniaestrains with resistance to specific antibiotics and use whole genome sequencing analysis for genotyping. As an exemplary case, we show that a single RNA polymerase beta subunit variant H526L (also frequently found in multidrug resistantMycobacterium tuberculosis) confers a 58-fold increase in CFE resistance to rifampicin. Overall, we describe a safe (i.e., non-living, non-pathogenic) platform suitable for studying an infectious disease model in a Containment Level 1 laboratory. Our CFE strategy is generalisable to laboratory and clinicalK. pneumoniaestrains and provides a new experimental tool to profile intracellular AMR variants. In conclusion, our CFE tool provides a significant advance towards understanding AMR and complements wider infectious disease studies.