1887

Abstract

is a prominent opportunistic nosocomial pathogen resistant to several classes of antibiotics. The major mechanism for fluoroquinolone resistance in various Gram-negative pathogens is active efflux. Our group previously identified SdeAB, a resistance-nodulation-cell division (RND) efflux pump complex, and a TolC-like outer-membrane protein (HasF), which together mediate energy-dependent fluoroquinolone efflux. In addition, a regulatory protein-encoding gene in the upstream region of was identified () and found to be 40 % homologous to MarA, an transcriptional regulator. To provide conclusive evidence as to the role of these components in , , and deletion mutants were constructed. Suicide vectors were created and introduced via triparental mating into UOC-67 (wild-type) and, for and , T-861 (clinical isolate). We have analysed these genetically altered strains using minimal inhibitory concentration (MIC) assays for a wide range of compounds (fluoroquinolones, SDS, novobiocin, ethidium bromide and chloramphenicol). Intracellular accumulation of a variety of fluoroquinolones was measured fluorospectroscopically. The , and knockout strains were consistently more susceptible to antibiotics than the parent strains, with the / double knockout strain showing the highest susceptibility. A marked increase in fluoroquinolone (ciprofloxacin) accumulation was observed for strains deficient in either the or genes when compared to the parental strains, with the highest ciprofloxacin accumulation observed for the / double knockout. Antibiotic accumulation assays for the knockout mutant strains performed in the presence of carbonyl cyanide -chlorophenylhydrazone (CCCP), a proton-motive-force inhibitor, demonstrated that SdeAB-mediated efflux is proton-motive-force dependent. Due to the comparable susceptibility of the and the individual knockouts, we conclude that HasF is the sole outer-membrane component of the SdeAB pump. In addition, MIC data for -deficient and overexpressing strains confirm that SdeR is an activator of and acts to enhance the overall multidrug resistance of .

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2008-02-01
2024-03-29
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