The long-term objectives of the proposed project are to understand the means by which bacteria circumvent the potent inhibitory activity of the fluoroquinolone antimicrobial agents. The fluoroquinolones are now used increasingly and have been effective in a wide range of human infections. With increasing use, bacterial resistance has been identified and limits efficacy in some clinical settings. Some bacteria develop quinolone resistance due to decreased drug permeation. In Escherichia coli quinolone resistance has been associated with mutations in novel genes which regulate porin outer membrane protein OmpF expression and interact with a newly identified system which actively exports quinolones from the cytoplasm. Analysis of these mutations and quinolone accumulation by bacteria is expected to provide further insight into the complex manner in which bacteria respond to their environment by altering their cell surface. Understanding these factors affecting drug permeation and their relationship to resistance might lead to means for limiting or bypassing such resistance.
The specific aims of the proposed project are (1) determination of the mans by which quinolone resistance mutations nfxB and cfxB reduce porin expression, (2) determination of other actions of nfxB and cfxB affecting quinolone resistance, (3) determination of the factors required for quinolone efflux from E. coli, (4) evaluation of factors affecting quinolone influx, and (5) characterization of quinolone accumulation and efflux in wildtype and resistant Staphylococcus aureus, an organism in which lacks an outer membrane. The methods to be used to accomplish these goals include (1) assays of the effect of nfxB and cfxB on ompF mRNA stability and on he expression of micF, a negative regulator of ompF, (2) assays of the effects of nfxB and cfxB on DNA supercoiling as measured by expression of lacZ from a supercoiling-dependent promoter and from a reporter plasmid, (3) assays of quinolone uptake by wild type and mutant bacteria, spheroplasts, and everted and right-side-out membrane vesicles, and (4) cloning of nfxB and cfxB.
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