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Antimicrobial Agents and Chemotherapy, August 2003, p. 2551-2557, Vol. 47, No. 8
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.8.2551-2557.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Haemophilus influenzae blaROB-1 Mutations in Hypermutagenic {Delta}ampC Escherichia coli Conferring Resistance to Cefotaxime and ß-Lactamase Inhibitors and Increased Susceptibility to Cefaclor

Juan-Carlos Galán,* María-Isabel Morosini, María-Rosario Baquero, Milagro Reig, and Fernando Baquero

Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Madrid, Spain

Received 17 January 2003/ Returned for modification 25 March 2003/ Accepted 17 May 2003

The clinical use of cefaclor has been shown to enrich Haemophilus influenzae populations harboring cefaclor-hydrolyzing ROB-1 ß-lactamase. Such a selective process may lead to the increased use of extended-spectrum cephalosporins or ß-lactams plus ß-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 ß-lactamase inhibitors, a plasmid harboring blaROB-1 was transformed into hypermutagenic strain Escherichia coli GB20 ({Delta}ampC 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 ß-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 ß-lactams plus ß-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 ß-lactamase toward molecular forms able to resist extended-spectrum cephalosporins or ß-lactamase inhibitors.

* Corresponding author. Mailing address: Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Ctra. de Colmenar, Km 9,1, 28034 Madrid, Spain. Phone: 34-91-336 8330. Fax: 34-91-336 8809. E-mail: jgalanm.hrc{at}salud.madrid.org.

Antimicrobial Agents and Chemotherapy, August 2003, p. 2551-2557, Vol. 47, No. 8
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.8.2551-2557.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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