(183z) Systems Level Study of Antibiotic Persistence Mechanisms

Antibiotic resistance is increasingly recognized as a global health crisis. One mechanism by which pathogens develop resistance is through repeated exposure to antibiotics, leading to the emergence of subpopulations known as persisters, which are tolerant to treatment. Despite the extensive study of persistence, the traditional, labor-intensive methods have impeded the discovery of new genes involved in this phenomenon. To address this, our project developed a high-throughput approach to identify transcription factors associated with persistence and uncover novel mechanisms. We employed a library of barcoded guide RNA knockdowns to simultaneously assess the impact of knocking down all Escherichia coli transcription factors on antibiotic survival. From a single pooled experiment, we generated antibiotic survival curves for all regulators and identified 14 transcriptional regulators that significantly affect persistence, including three regulators not previously linked to persistence. To validate our pooled data, individual strains were subjected to standard persistence assays, demonstrating consistency with previously published results. Individual tested strains effectively reproduced pooled data as well as correlated with knockout strains. This high-throughput method enables the rapid screening of hundreds of regulators to elucidate potential survival strategies of pathogens under treatment. Additionally, it holds promise for broader applications in medical and bioproduction fields where survival mechanisms are of interest. This approach not only accelerates the discovery of persistence-related genes but also expands our understanding of bacterial survival strategies, potentially guiding the development of more effective therapeutic interventions.