Eller, K. A. | Search bioRxiv

bioRxiv2019

Facile Accelerated Specific Therapeutic (FAST) Platform to Counter Multidrug-Resistance Bacteria

Multidrug-resistant (MDR) bacteria pose a grave concern to global health. This problem is further aggravated by a lack of new and effective antibiotics and countermeasure platforms that can sustain the creation of novel antimicrobials in the wake of new outbreaks or evolution of resistance to antibiotics. To address this, we have developed a Facile Accelerated Specific Therapeutic (FAST) platform that can develop effective therapies against MDR bacteria within a week. Our FAST platform combines four essential modules- design, build, test, and delivery-of drug development cycle. The design module comprises a bioinformatics toolbox that predicts sequence-specific peptide nucleic acids (PNAs) that target non-traditional pathways and genes of bacteria in minutes. The build module constitutes in-situ synthesis and validation of selected PNAs in less than four days and efficacy testing within a day. As a proof of concept, these PNAs were tested against MDR clinical isolates. Here we tested Enterobacteriaceae including carbapenem-resistant Escherichia coli, extended-spectrum beta-lactamase (ESBL) Klebsiella pneumoniae, New Delhi Metallo-beta-lactamase-1 carrying Klebsiella pneumoniae and MDR Salmonella enterica. PNAs showed significant growth inhibition for 82% of treatments, with nearly 18% of the treatments leading to more than 97% decrease. Further, these PNAs are capable of potentiating antibiotic activity in the clinical isolates despite presence of cognate resistance genes. Finally, FAST offers a novel delivery approach to overcome limited transport of PNAs into mammalian cells to clear intracellular infections. This method relies on repurposing the bacterial Type III secretion system in conjunction with a kill switch that is effective at eliminating 99.6% of an intracellular Salmonella infection in human epithelial cells. Our findings demonstrate the potential of the FAST platform in treating MDR bacteria in a rapid and effective manner.

bioengineering↗

bioRxiv2019

Potentiating antibiotic treatment using fitness-neutral gene expression perturbations

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.

synthetic biology↗