Haemophilus influenzae bla ROB-1 Mutations in Hypermutagenic Δ ampC Escherichia coli Conferring Resistance to Cefotaxime and β-Lactamase Inhibitors and Increased Susceptibility to Cefaclor

Abstract

ABSTRACT 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 bla ROB-1 mutations, if any, could confer resistance to extended-spectrum cephalosporins and/or to β-lactamase inhibitors, a plasmid harboring bla ROB-1 was transformed into hypermutagenic strain Escherichia coli GB20 (Δ ampC mutS ::Tn 10 ), 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 β-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 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 bla ROB-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.