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.