Potentiating antibiotic treatment using fitness-neutral gene expression perturbations

Abstract

The rapid proliferation of multidrug-resistant (MDR) bacteria poses a critical threat to human health, for which new antimicrobial strategies are desperately needed. Here we outline a strategy for combating bacterial infections by administering fitness neutral gene expression perturbations as co-therapies to potentiate antibiotic lethality. We systematically explored the fitness of 270 gene knockout-drug combinations in Escherichia coli, identifying 114 synergistic interactions. Genes revealed in this screen were subsequently perturbed at the transcriptome level via multiplexed CRISPR-dCas9 interference to induce antibiotic synergy. These perturbations successfully sensitized E. coli to antibiotic treatment without imposing a separate fitness cost. We next administered these fitness neutral gene perturbations as co-therapies to potentiate antibiotic killing of Salmonella enterica in intracellular infections of HeLa epithelial cells, demonstrating therapeutic applicability. Finally, we utilized these results to design peptide nucleic acid (PNA) co-therapies for targeted gene expression reduction in four MDR, clinically isolated bacteria. Two isolates of Klebsiella pneumoniae and E. coli were each exposed to PNAs targeting homologs of the genes csgD, fnr, recA and acrA in the presence of sub-minimal inhibitory concentrations of trimethoprim. We successfully increased each strains susceptibility to trimethoprim treatment and identified eight cases in which re-sensitization occurred without a direct fitness impact of the PNA on MDR strains. Our results highlight a promising approach for combating MDR bacteria which could extend the utility of our current antibiotic arsenal.