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Proliferation of multidrug-resistant (MDR) bacteria poses a threat to human health, requiring new strategies. Here we propose using fitness neutral gene expression perturbations to potentiate antibiotics. We systematically explored 270 gene knockout-antibiotic combinations inEscherichia coli, identifying 90 synergistic interactions. Identified gene targets were subsequently tested for antibiotic synergy on the transcriptomic level via multiplexed CRISPR-dCas9 and showed successful sensitization ofE. coliwithout a separate fitness cost. These fitness neutral gene perturbations worked as co-therapies in reducing aSalmonella entericaintracellular infection in HeLa. Finally, these results informed the design of four antisense peptide nucleic acid (PNA) co-therapies,csgD,fnr,recAandacrA, against four MDR, clinically isolated bacteria. PNA combined with sub-minimal inhibitory concentrations of trimethoprim against two isolates ofKlebsiella pneumoniaeandE. colishowed three cases of re-sensitization with minimal fitness impacts. Our results highlight a promising approach for extending the utility of current antibiotics.

Multidrug-resistant (MDR) bacteria pose a grave concern to global health, which is perpetuated by a lack of new treatments and countermeasure platforms to combat outbreaks or antibiotic resistance. To address this, we have developed a Facile Accelerated Specific Therapeutic (FAST) platform that can develop effective peptide nucleic acid (PNA) therapies against MDR bacteria within a week. Our FAST platform uses a bioinformatics toolbox to design sequence-specific PNAs targeting non-traditional pathways/genes of bacteria, then performs in-situ synthesis, validation, and efficacy testing of selected PNAs. As a proof of concept, these PNAs were tested against five MDR clinical isolates: carbapenem-resistantEscherichia coli, extended-spectrum beta-lactamaseKlebsiella pneumoniae, New Delhi Metallo-beta-lactamase-1 carryingKlebsiella pneumoniae, and MDRSalmonella enterica. PNAs showed significant growth inhibition for 82% of treatments, with nearly 18% of treatments leading to greater than 97% decrease. Further, these PNAs are capable of potentiating antibiotic activity in the clinical isolates despite presence of cognate resistance genes. Finally, the FAST platform offers a novel delivery approach to overcome limited transport of PNAs into mammalian cells by repurposing the bacterial Type III secretion system in conjunction with a kill switch that is effective at eliminating 99.6% of an intracellularSalmonellainfection in human epithelial cells.