In Vitro Prediction of the Evolution of the GES - 1 - Lactamase 1 Hydrolytic Activity

22 Resistance to ß-lactams is constantly increasing, due to the emergence of totally new 23 enzymes, but also to the evolution of pre-existing ß-lactamases. GES-1 is a clinically24 relevant extended-spectrum -lactamase (ESBL) hydrolyzing penicillins and broad25 spectrum cephalosporins, but sparing monobactams and carbapenems. However, 26 several GES-1 variants (i.e. GES-2 and GES-5) previously identified among clinical 27 isolates display an extended spectrum of activity toward carbapenems. To study the 28 evolution potential of the GES-1 -lactamase, this enzyme was submitted to in-vitro 29 directed evolution, with selection on increasing concentrations of the cephalosporin 30 cefotaxime, the monobactam aztreonam, or the carbapenem imipenem. The highest 31 resistance levels were conferred by the combination of up to four substitutions. The 32 A6T, E104K, G243A variant selected on cefotaxime, and the A6T, E104K, T237A, 33 G243A variant selected on aztreonam, conferred high resistance to cefotaxime, 34 ceftazidime, and aztreonam. Conversely, the A6T, G170S variant selected on imipenem 35 conferred high resistance to imipenem and cefoxitin. Noteworthy, the A6T substitution 36 involved in higher MICs for all ß-lactams is located in the leader peptide of the GES 37 enzyme, therefore not present in the mature protein. Acquired cross resistance was not 38 2 ht tp :// do c. re ro .c h observed since selection with CTX or ATM did not select for resistance to IPM and vice 39 versa. Here we demonstrated that -lactamase GES-1 exhibits peculiar properties with a 40 significant potential to gain activity toward broad-spectrum cephalosporins, 41 monobactams, and carbapenems. 42