New mutations in ADC-type β-lactamases from Acinetobacter spp. affect cefoxitin and ceftazidime hydrolysis.
OBJECTIVE
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.
Servicio de Microbiología-INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain.
Astrid
A
Pérez
Servicio de Microbiología-INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain.
Francisco José
FJ
Pérez-Llarena
Servicio de Microbiología-INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain.
Patricia
P
García
Centre d'Ingénierie des Protéines, Université de Liège, Liège, Belgium.
Frédéric
F
Kerff
Servicio de Microbiología-INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain.
Alejandro
A
Beceiro
Centre d'Ingénierie des Protéines, Université de Liège, Liège, Belgium.
Moreno
M
Galleni
Servicio de Microbiología-INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain german.bou.arevalo@sergas.es german.bou@usc.es.
Germán
G
Bou
D000150:Acinetobacter / Q000187:drug effects / Q000201:enzymology*; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D002440:Cefoxitin / Q000378:metabolism*; D002442:Ceftazidime / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006868:Hydrolysis; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D050505:Mutant Proteins; D011994:Recombinant Proteins; D002440:Cefoxitin; D002442:Ceftazidime; D001618:beta-Lactamases
ADC-type class C β-lactamases; Acinetobacter; structure–function