458: Improved Genetic Engineering of Gram-Negative Bacteria for Bioluminescent Assays and Antibiotic Testing

Voiceover

Presenter(s)

Ihika Lagisetty

Abstract or Description

The resistance of bacteria to antibiotics is a growing worldwide public health threat. Half of the bacteria considered by the CDC as urgent or serious threats are gastrointestinal pathogens. Sensitivity to antibiotics is conventionally assessed by the Kirby-Bauer method where bacteria are grown on agar plates with antibiotic-soaked wavers and the size of the bacteria-free zone surrounding each waver indicates each antibiotic's effectiveness. However, we want to analyze the antibacterial efficacy of candidate antibiotics in real-time in vitro and in vivo using bioluminescence imaging (BLI). However, few bacterial species, e.g. some Vibrio cholerae strains, have an endogenous light-emitting luciferase enzyme, whereas most other species need to be genetically engineered. For this, we are using a mobile genetic element, the transposon Tn7, to insert a set of genes (lux operon) into the bacterial genome that encode a bacterial luciferase and the substrate synthesizing enzymes. The Tn7 plasmid, unfortunately, can only be selected for, making the removal of the plasmid backbone after transposition time-consuming. We are currently replacing the antibiotic selection marker, beta lactamase, with the tetracycline resistance marker, which can be both selected for and against. This modified plasmid will permit us to label clinical isolates of enteropathogenic E. coli (EPEC) and assess bacterial survival in response to various plant-derived antibiotics. Finally, in preparation for clinical trials we will assess their treatment efficacy in a mouse model of gastrointestinal infection with bioluminescent EPEC that we will also monitor by BLI.