Abstracts

Antimicrobial resistance is on the rise, set to surpass the major causes of death for humans by 2050. Two bacterial species, Escherichia coli and Pseudomonas aeruginosa, are becoming highly antibiotic resistant, being important pathogens in nosocomial infections. The aim of our project is to generate reporter bacterial strains with genomic integration of genes for expression of a luciferase (either CBG2 or NanoLuc) and a fluorescent tag (either miniSOG or mKate2) to be used as a sensitive method in vitro and in vivo for antimicrobials efficacy assessment. Plasmids containing either reporter genes were generated using Gibson Assembly. The promoter J23119 was chosen to enhance the expression of both proteins in the final constructs. Plasmids containing the homology arms for the 16s locus of the bacteria will be used as donor DNA plasmid for the CRISPR/Cas9 gene editing technology. Lambda/Red recombination strategy will facilitate the integration in both species (ATCC25922/Niessle 1917 strains for E. coli and ATCC27853/PA01 strains for P. aeruginosa). CBG2/NanoLuc expressing bacteria have been imaged using the IVIS Spectrum Imager (PerkinElmer) after incubation of D-Luciferin, Naphtyl-Luciferin (CBG2) and Furimazine (NanoLuc). MiniSOG/mKate2 fluorescence has been visualized using the microscopes DM5500 B and SP8 DLS (Leica). Our preliminary results demonstrate that a series of constructs for the dual expression of CBG2/NanoLuc and miniSOG/mKate2 has been successfully assembled in gram negative bacteria for CRISPR/Cas9 integration. All reporter proteins were correctly expressed and bacteria may be applied for different assays for the testing of antimicrobials in vitro and in vivo using optical readouts.