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See detailNew mutations in ADC-type beta-lactamases from Acinetobacter spp. affect cefoxitin and ceftazidime hydrolysis.
Perez, Astrid; Perez-Llarena, Francisco Jose; Garcia, Patricia et al

in The Journal of antimicrobial chemotherapy (2014)

OBJECTIVES: Two natural variants of ADC-type beta-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 ... [more ▼]

OBJECTIVES: Two natural variants of ADC-type beta-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 beta-lactam antibiotics in this family of beta-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 beta-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 beta-lactamases, little is known about ceftazidime and cefoxitin hydrolysis in this family of beta-lactamases. Hydrolysis of these antibiotics appears to be positively and negatively affected, respectively, by the Q163K, P167S, D242G and G342R amino acid replacements. [less ▲]

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See detailCharacterization of the new AmpC beta-lactamase FOX-8 reveals a single mutation, Phe313Leu, located in the R2 loop that affects ceftazidime hydrolysis.
Perez-Llarena, Francisco Jose; Kerff, Frédéric ULg; Zamorano, Laura et al

in Antimicrobial agents and chemotherapy (2013), 57(10), 5158-61

A novel class C beta-lactamase (FOX-8) was isolated from a clinical strain of Escherichia coli. The FOX-8 enzyme possessed a unique substitution (Phe313Leu) compared to FOX-3. Isogenic E. coli strains ... [more ▼]

A novel class C beta-lactamase (FOX-8) was isolated from a clinical strain of Escherichia coli. The FOX-8 enzyme possessed a unique substitution (Phe313Leu) compared to FOX-3. Isogenic E. coli strains carrying FOX-8 showed an 8-fold reduction in resistance to ceftazidime relative to FOX-3. In a kinetic analysis, FOX-8 displayed a 33-fold reduction in kcat/Km for ceftazidime compared to FOX-3. In the FOX family of beta-lactamases, the Phe313 residue located in the R2 loop affects ceftazidime hydrolysis and alters the phenotype of E. coli strains carrying this variant. [less ▲]

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