title: BmeRABC5 is a multidrug efflux system that can confer metronidazole resistance in Bacteroides fragilis.
abstract: The RND-family efflux pump gene bmeB5 was previously shown to be overexpressed in metronidazole-resistant laboratory mutants of Bacteroides fragilis. In the present study, we characterized the bmeABC5 genes and an upstream putative TetR-family regulator gene (bmeR5). bmeR5 (645 bp) was located 51 bp upstream of bmeA5 and encoded a 24.9-kDa protein. Deletant strains lacking bmeB5 or bmeR5 were constructed from a wild-type B. fragilis strain ADB77. Strain antimicrobial susceptibility was determined and gene expression was quantified. bmeR5 was overexpressed in Escherichia coli using a 6x-His tag system; BmeR5-His6 was isolated from inclusion bodies and its binding to bmeABC5 promoter regions was determined. BmeR5-His6 bound specifically to the bmeR5-bmeC5 intergenic region (IT1). Deletion of bmeR5 (ADB77DeltabmeR5) resulted in a significant (p < 0.05) increase in expression of bmeA5, bmeB5, and bmeC5, and > two-fold increase in minimum inhibitory concentrations (MICs) of ampicillin, cefoxitin, cefoperazone, ciprofloxacin, imipenem, metronidazole, ethidium bromide, and sodium dodecyl sulfate (SDS). MICs were reduced by the efflux pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The MICs of ampicillin, cefoperazone, metronidazole, and SDS were reduced by approximately two-fold in ADB77DeltabmeB5. A multidrug (metronidazole)-resistant, nim-negative B. fragilis clinical isolate overexpressed bmeABC5 genes, had a G-->T point mutation in IT1, and significantly reduced binding to BmeR5-His6. These data demonstrate that BmeR5 is a local repressor of bmeABC5 expression and that mutations in IT1 can lead to a derepression and resistance to multiple antimicrobial agents, including metronidazole.
authors.affiliation: Greater Los Angeles Veterans Administration Healthcare Systems, Los Angeles, CA 90073, USA. lilskil@ucla.edu
authors.forename: Lilian
authors.initials: L
authors.lastname: Pumbwe
authors.forename: Abraham
authors.initials: A
authors.lastname: Chang
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Smith
authors.forename: Hannah M
authors.initials: HM
authors.lastname: Wexler
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D001441:Bacteroides fragilis / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008795:Metronidazole / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D008795:Metronidazole