title: Haemophilus influenzae bla(ROB-1) mutations in hypermutagenic deltaampC Escherichia coli conferring resistance to cefotaxime and beta-lactamase inhibitors and increased susceptibility to cefaclor.
abstract: The clinical use of cefaclor has been shown to enrich Haemophilus influenzae populations harboring cefaclor-hydrolyzing ROB-1 beta-lactamase. Such a selective process may lead to the increased use of extended-spectrum cephalosporins or beta-lactams plus beta-lactamase inhibitors and, eventually, resistance to these agents, which has not previously been observed in H. influenzae. In order to establish which bla(ROB-1) mutations, if any, could confer resistance to extended-spectrum cephalosporins and/or to beta-lactamase inhibitors, a plasmid harboring bla(ROB-1) was transformed into hypermutagenic strain Escherichia coli GB20 (DeltaampC mutS::Tn10), and this construct was used in place of H. influenzae bla(ROB-1). Strain GB20 with the cloned gene was submitted to serial passages in tubes containing broth with increasing concentrations of selected beta-lactams (cefotaxime or amoxicillin-clavulanate). Different mutations in the bla(ROB-1) gene were obtained during the passages in the presence of the different concentrations of the selective agents. Mutants resistant to extended-spectrum cephalosporins harbored either the Leu169-->Ser169 or the Arg164-->Trp164 substitution or the double amino acid change Arg164-->Trp164 and Ala237-->Thr237. ROB-1 mutants that were resistant to beta-lactams plus beta-lactamase inhibitors and that harbored the Arg244-->Cys244 or the Ser130-->Gly130 replacement were also obtained. The cefaclor-hydrolyzing efficiencies of the ROB-1 variants were strongly decreased in all mutants, suggesting that if bla(ROB-1) mutants were selected by cefaclor, this drug would prevent the further evolution of this beta-lactamase toward molecular forms able to resist extended-spectrum cephalosporins or beta-lactamase inhibitors.
authors.affiliation: Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Madrid, Spain. jgalanm.hrc@salud.madrid.org
authors.forename: Juan-Carlos
authors.initials: JC
authors.lastname: Galán
authors.forename: María-Isabel
authors.initials: MI
authors.lastname: Morosini
authors.forename: María-Rosario
authors.initials: MR
authors.lastname: Baquero
authors.forename: Milagro
authors.initials: M
authors.lastname: Reig
authors.forename: Fernando
authors.initials: F
authors.lastname: Baquero
mesh_terms: D019980:Amoxicillin-Potassium Clavulanate Combination / Q000494:pharmacology; D001426:Bacterial Proteins; D002433:Cefaclor / Q000494:pharmacology*; D002439:Cefotaxime / Q000494:pharmacology*; D018550:Cephalosporin Resistance; D002511:Cephalosporins / Q000494:pharmacology*; D004359:Drug Therapy, Combination / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D006193:Haemophilus influenzae / Q000235:genetics*; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D009154:Mutation / Q000502:physiology*; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002511:Cephalosporins; D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D002433:Cefaclor; D019980:Amoxicillin-Potassium Clavulanate Combination; C048795:AmpC beta-lactamases; D001618:beta-Lactamases; C477296:beta-lactamase bla(ROB-1), Haemophilus influenzae; D002439:Cefotaxime