Haemophilus influenzae blaROB-1 Mutations in Hypermutagenic ΔampC Escherichia coli Conferring Resistance to Cefotaxime and β-Lactamase Inhibitors and Increased Susceptibility to Cefaclor

The clinical use of cefaclor has been shown to enrich Haemophilus influenzae populations harboring cefaclor-hydrolyzing ROB-1 β-lactamase. Such a selective process may lead to the increased use of extended-spectrum cephalosporins or β-lactams plus β-lactamase inhibitors and, eventually, resistance to these agents, which has not previously been observed in H. influenzae. In order to establish which blaROB-1 mutations, if any, could confer resistance to extended-spectrum cephalosporins and/or to β-lactamase inhibitors, a plasmid harboring blaROB-1 was transformed into hypermutagenic strain Escherichia coli GB20 (ΔampC mutS::Tn10), and this construct was used in place of H. influenzae blaROB-1. Strain GB20 with the cloned gene was submitted to serial passages in tubes containing broth with increasing concentrations of selected β-lactams (cefotaxime or amoxicillin-clavulanate). Different mutations in the blaROB-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 β-lactams plus β-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 blaROB-1 mutants were selected by cefaclor, this drug would prevent the further evolution of this β-lactamase toward molecular forms able to resist extended-spectrum cephalosporins or β-lactamase inhibitors.