Identification of Determinants that Allow Maintenance of High-Level Fluoroquinolone Resistance inAcinetobacter baumannii

Abstract

AbstractAcinetobacter baumanniiis associated with multidrug resistant (MDR) infections in healthcare settings, with fluoroquinolones such as ciprofloxacin being currently ineffective. Clinical isolates largely harbor mutations in the GyrA and TopoIV fluoroquinolone targets, as well as mutations that increase expression of drug resistance-nodulation-division (RND) efflux pumps. Factors critical for maintaining fitness levels of pump overproducers are uncharacterized despite their prevalence in clinical isolates, so proteins that contribute to fitness of FQR strains overexpressing three known RND systems were identified using high-density insertion mutagenesis. Overproduction of the AdeFGH efflux pump caused hypersensitization to defects in outer membrane homeostatic regulation, including lesions that reduced peptidoglycan recycling and LOS biosynthesis, and interfered with outer membrane lipid asymmetry driven by the Mla system. In contrast, AdeAB pump overproduction, which does not affect the outer membrane pump component, was relatively tolerant to loss of these functions, consistent with outer membrane protein overproduction being the primary disruptive component. Surprisingly, overproduction of proton-transporting efflux pumps had little impact on cytosolic pH, consistent with a compensatory response to pump activity. The most striking transcriptional changes were associated with AdeFGH pump overproduction, resulting in activation of the phenylacetate (PAA) degradation regulon. Disruption of the PAA pathway resulted in cytosolic acidification and defective expression of genes involved in protection from peroxide stress. These results indicate that the RND outer membrane protein overproduction is compensated by cytoplasmic buffering and maintenance of outer membrane integrity inA. baumanniito facilitate fitness of FQR isolates.