Identification of essential genes that support fitness of Acinetobacter baumannii efflux pump overproducers in the presence of fluoroquinolone

We designed a 12,000 sgRNA library and introduced it into four A. baumannii strains: WT (ATCC 17978UN) and three high-level fluoroquinolone resistant strains which harbor mutations in the GyrA and TopoIV fluoroquinolone targets as well as mutations that increase expression of drug resistance-nodulation-division (RND) efflux pumps (gyrAparCadeS (GPS), gyrAparCadeN (GPN), and gyrAparCadeL (GPL)). These libraries were cultured for 7-8 generations (or 14-16 generations) in the absence of inducer, in the presence of inducer (50, 100, or 200ng/mL of ATC) and absence of drug, or in the presence of inducer with comparable growth inhibiting levels of ciprofloxacin to evaluate the efficacy of our CRISPRi screening strategy and to identify hypomorphic mutants that significantly impact the fitness and drug sensitivity of the pump overproducers.