(191m) Fluroquinolone Mediated Mutagenesis and Its Effect on Drug Tolerance/Resistance

Antibiotic resistance is a global health crisis that poses a significant threat to the medical progress achieved over the past century. Bacterial cells are capable of rapidly evolving resistance to antibiotics, in part due to the error-prone DNA repair mechanisms, which are activated in response to bacterial DNA damage. While these DNA repair mechanisms are a fundamental survival strategy that maintains bacterial genomic integrity, its mutagenic processes can lead to the formation of antibiotic-resistant cells. Persister cells further exacerbate this global problem. These cells are phenotypic variants that exist in a non-heritable, reversible, antibiotic-tolerant state triggered by stochastic and/or deterministic factors associated with stress response. Although persistence and resistance are two distinct phenomena, they are interrelated, as persisters can serve as reservoirs for resistance development. Therefore, a thorough understanding of mutagenic mechanisms is necessary to develop effective clinical treatment strategies to combat the global health crisis of drug tolerance/resistance.

Here, our goal was to map and control cellular repair-mediated mutagenesis and persistence. We focused on the quinolone class of antibiotics, which are widely used to treat bacterial infections but are also known to drive antibiotic resistance. A two-step high-throughput screening strategy was implemented using the second-generation fluoroquinolone, ciprofloxacin. In the first method, a fluorescent reporter plasmid library encompassing the promoters of DNA repair genes was used to identify active repair mechanisms following ciprofloxacin treatment. For a more in-depth analysis, a second screening step was performed using a single deletion library targeting genes involved in potential DNA repair mechanisms, including recA-mediated SOS response, nucleotide excision repair, base excision repair and mismatch repair. This strategy allowed us to examine both the extent of mutagenesis and persistence, revealing key DNA repair genes —particularly those involved in homologous recombination, such as recB and ruvC— whose deletion led to a significant reduction in mutagenesis and persistence. These deletions also resulted in increased antibiotic susceptibility and a transient loss of culturability. These results were further verified employing different generations of quinolones, levofloxacin (third generation), and moxifloxacin (fourth generation) and across different variants of E. coli, including a uropathogenic strain (UPEC). Overall, this study highlights the key repair pathways along with key genes that play a crucial role in both persistence and mutagenesis, and hence can be used as a potential therapeutic target against antibiotic tolerance/resistance.