title: In vitro prediction of the evolution of GES-1 β-lactamase hydrolytic activity.
abstract: Resistance to β-lactams is constantly increasing due to the emergence of totally new enzymes but also to the evolution of preexisting β-lactamases. GES-1 is a clinically relevant extended-spectrum β-lactamase (ESBL) that hydrolyzes penicillins and broad-spectrum cephalosporins but spares monobactams and carbapenems. However, several GES-1 variants (i.e., GES-2 and GES-5) previously identified among clinical isolates display an extended spectrum of activity toward carbapenems. To study the evolution potential of the GES-1 β-lactamase, this enzyme was submitted to in vitro-directed evolution, with selection on increasing concentrations of the cephalosporin cefotaxime, the monobactam aztreonam, or the carbapenem imipenem. The highest resistance levels were conferred by a combination of up to four substitutions. The A6T-E104K-G243A variant selected on cefotaxime and the A6T-E104K-T237A-G243A variant selected on aztreonam conferred high resistance to cefotaxime, ceftazidime, and aztreonam. Conversely, the A6T-G170S variant selected on imipenem conferred high resistance to imipenem and cefoxitin. Of note, the A6T substitution involved in higher MICs for all β-lactams is located in the leader peptide of the GES enzyme and therefore is not present in the mature protein. Acquired cross-resistance was not observed, since selection with cefotaxime or aztreonam did not select for resistance to imipenem, and vice versa. Here, we demonstrate that the β-lactamase GES-1 exhibits peculiar properties, with a significant potential to gain activity against broad-spectrum cephalosporins, monobactams, and carbapenems.
authors.affiliation: Medical and Molecular Microbiology Unit, Department of Medicine, Faculty of Science, University of Fribourg, Fribourg, Switzerland.
authors.forename: Séverine
authors.initials: S
authors.lastname: Bontron
authors.affiliation: Medical and Molecular Microbiology Unit, Department of Medicine, Faculty of Science, University of Fribourg, Fribourg, Switzerland INSERM U914 Emerging Resistance to Antibiotics, Faculté de Médecine et Université Paris Sud, Le Kremlin-Bicêtre, France laurent.poirel@unifr.ch.
authors.forename: Laurent
authors.initials: L
authors.lastname: Poirel
authors.affiliation: Medical and Molecular Microbiology Unit, Department of Medicine, Faculty of Science, University of Fribourg, Fribourg, Switzerland INSERM U914 Emerging Resistance to Antibiotics, Faculté de Médecine et Université Paris Sud, Le Kremlin-Bicêtre, France HFR-Hôpital Cantonal de Fribourg, Fribourg, Switzerland.
authors.forename: Patrice
authors.initials: P
authors.lastname: Nordmann
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D006868:Hydrolysis; D008826:Microbial Sensitivity Tests / Q000379:methods; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism*; D047090:beta-Lactams / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D047090:beta-Lactams; C405902:GES-1 beta-lactamase; D001618:beta-Lactamases