Astrid Pérez1, Francisco José Pérez-Llarena1, Patricia García1, Frédéric Kerff2, Alejandro Beceiro1, Moreno Galleni2, Germán Bou3. 1. Servicio de Microbiología-INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain. 2. Centre d'Ingénierie des Protéines, Université de Liège, Liège, Belgium. 3. Servicio de Microbiología-INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain german.bou.arevalo@sergas.es german.bou@usc.es.
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.
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.
Authors: Massimiliano Marazzato; Daniela Scribano; Meysam Sarshar; Francesca Brunetti; Silvia Fillo; Antonella Fortunato; Florigio Lista; Anna Teresa Palamara; Carlo Zagaglia; Cecilia Ambrosi Journal: Int J Environ Res Public Health Date: 2022-03-01 Impact factor: 3.390