2024 AIChE Annual Meeting

Riboswitch Engineering : Tracking Levofloxacin Accumulation and Oxidative Damage in Single Cells Using Biosensors

Background & Significance: Antibiotic treatment failure occurs when bacterial cells survive treatment, which can lead to bacterial infection regrowth after the antibiotics are removed and the surviving cells begin to propagate. This phenomenon can be detrimental for a patient, as they may improve during treatment only for infections to worsen again as these surviving persisters regrow. Persisters are different from resistant cells. Persisters are genetically identical to other cells in the population that die during antibiotic treatment, but they can change their phenotypic responses to tolerate drugs. By comparison, resistant cells are driven by evolution and caused by genetic mutations that allow them to fight against the antibiotic. Single-cell techniques are needed to identify what is happening to these persister cells during antibiotic treatment.

Methods: In this project, we are engineering two biosensors, one for checking oxidative damage on 8-oxoguanine (8-oxo-G), and another to check for accumulation of Levofloxacin (Levo), our antibiotic of interest. We are generating different designs of the biosensors by site-directed mutagenesis (SDM) and molecular cloning. We are assessing the activity and ligand-sensing ability of these sensors by fluorescence spectroscopy and flow cytometry.

Results: I generated some variants of the original biosensors by SDM. Many of the variants can generate a fluorescent signal, but they can also do so in the absence of the ligands. I am optimizing the sensors to ensure that they are only fluorescent when the ligands are present.

Conclusions: Using these two biosensors in bacteria will enable us to determine if bacterial persisters are experiencing damage from the Levo antibiotic, and to see if Levo is accumulating in the persisters and cells that die.