Pérez, AstridPérez-Llarena, Francisco J.García, PatriciaKerff, FrédéricBeceiro Casas, AlejandroGalleni, MorenoBou, Germán2026-09-222026-09-222014-05-20Pérez A, Pérez-Llarena FJ, García P, Kerff F, Beceiro A, Galleni M, Bou G. New mutations in ADC-type β-lactamases from Acinetobacter spp. affect cefoxitin and ceftazidime hydrolysis. J Antimicrob Chemother. 2014 Sep;69(9):2407-11.1460-2091https://hdl.handle.net/2183/49358This is a pre-copyedited, author-produced version of an article accepted for publication in Journal of Journal of Antimicrobial Chemotherapy following peer review. The version of record is available online at: https://doi.org/10.1093/JAC/DKU163.[Abstract] Objectives: Two natural variants of ADC-type β-lactamases of Acinetobacter spp., ADC-1 and ADC-5, differ by nine mutations in their protein sequence. ADC-5 hydrolyses cefoxitin better than ADC-1 and the opposite is true for ceftazidime. We produced single and combined mutations in ADC-5 and characterized the variants microbiologically and biochemically to determine which amino acid residues are involved in the hydrolysis of β-lactam antibiotics in this family of β-lactamases. Methods: Site-directed mutagenesis, with blaADC-5 as a source of DNA, was used to generate nine single mutated and three combined mutated enzymes. The proteins (wild-type and derivatives) were then expressed in isogenic conditions in Escherichia coli. MICs of β-lactams were determined using Etest strips. ADC-1, ADC-5, ADC-5-P167S and ADC-5-P167S/D242G/Q163K/G342R were also purified and the kinetic parameters determined for ceftazidime, cefoxitin, cefalotin and ampicillin. Results: Single mutations did not significantly convert the hydrolysis spectrum of the ADC-5 enzyme into that of the ADC-1 enzyme, although among all studied mutants only the quadruple mutant (ADC-5-P167S/D242G/Q163K/G342R) displayed microbiological and biochemical properties consistent with those of ADC-1. Conclusions: Although some single mutations are known to affect cefepime hydrolysis in ADC-type β-lactamases, little is known about ceftazidime and cefoxitin hydrolysis in this family of β-lactamases. Hydrolysis of these antibiotics appears to be positively and negatively affected, respectively, by the Q163K, P167S, D242G and G342R amino acid replacements.eng© 2014. Published by Oxford University Press. All rights reserved.ADC-type class C β-lactamasesAcinetobacterStructure–functionjournal articleopen access10.1093/JAC/DKU163