title: Genetic and kinetic characterization of the novel AmpC β-lactamases DHA-6 and DHA-7.
abstract: During a Spanish surveillance study, two natural variants of DHA β-lactamases, DHA-6 and DHA-7, were found, with the replacements Ala226Thr and Phe322Ser, respectively, with respect to DHA-1. The DHA-6 and DHA-7 enzymes were isolated from Escherichia coli and Enterobacter cloacae clinical isolates, respectively. The aim of this study was to genetically, microbiologically, and biochemically characterize the DHA-6 and DHA-7 β-lactamases. The blaDHA-6 and blaDHA-7 genes were located in the I1 and HI2 incompatibility group plasmids of 87.3 and 310.4 kb, respectively. The genetic contexts of blaDHA-6 and blaDHA-7 were similar to that already described for the blaDHA-1 gene and included the qnrB4 and aadA genes. The MICs for cephalothin, aztreonam, cefotaxime, and ceftazidime were 8- to 32-fold lower for DHA-6 than for DHA-1 or DHA-7 expressed in the same isogenic E. coli TG1 strain. Interestingly, the MIC for cefoxitin was higher in the DHA-6-expressing transformant than in DHA-1 or DHA-7. Biochemical studies with pure β-lactamases revealed slightly lower catalytic efficiencies of DHA-6 against cephalothin, ceftazidime, and cefotaxime than those of DHA-1 and DHA-7. To understand this behavior, stability experiments were carried out and showed that the DHA-6 protein displayed significantly higher stability than the DHA-1 and DHA-7 enzymes. The proximity of Thr226 to the N terminus in the tertiary protein structure in DHA-6 may promote this stabilization and, consequently, may induce a slight reduction in the dynamic of this enzyme that primarily affects the hydrolysis of some of the bulkiest antibiotics.
authors.affiliation: Servicio de Microbiología-INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain.
authors.forename: Francisco José
authors.initials: FJ
authors.lastname: Pérez-Llarena
authors.affiliation: Servicio de Microbiología, Hospital Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Zamorano
authors.affiliation: Centre d'Ingénierie des Protéines, Université de Liège, Liège, Belgium.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Kerff
authors.affiliation: Servicio de Microbiología-INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Beceiro
authors.affiliation: Servicio de Microbiología-INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: García
authors.affiliation: Servicio de Microbiología, Hospital de la Santa Creu i Sant Pau/IIB-Sant Pau, Universitat Autónoma de Barcelona, Barcelona, Spain.
authors.forename: Elisenda
authors.initials: E
authors.lastname: Miró
authors.affiliation: Servicio de Microbiología, Hospital Vall d'Hebron, Universitat Autónoma de Barcelona, Barcelona, Spain.
authors.forename: Nieves
authors.initials: N
authors.lastname: Larrosa
authors.affiliation: Servicio de Microbiología, Hospital Universitari Joan XXIII de Tarragona, Tarragona, Spain.
authors.forename: Frederic
authors.initials: F
authors.lastname: Gómez-Bertomeu
authors.affiliation: Servicio de Microbiología-INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain.
authors.forename: José Antonio
authors.initials: JA
authors.lastname: Méndez
authors.affiliation: Servicio de Microbiología, Hospital Vall d'Hebron, Universitat Autónoma de Barcelona, Barcelona, Spain.
authors.forename: Juan José
authors.initials: JJ
authors.lastname: González-López
authors.affiliation: Servicio de Microbiología, Hospital Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Oliver
authors.affiliation: Centre d'Ingénierie des Protéines, Université de Liège, Liège, Belgium.
authors.forename: Moreno
authors.initials: M
authors.lastname: Galleni
authors.affiliation: Servicio de Microbiología, Hospital de la Santa Creu i Sant Pau/IIB-Sant Pau, Universitat Autónoma de Barcelona, Barcelona, Spain.
authors.forename: Ferran
authors.initials: F
authors.lastname: Navarro
authors.affiliation: Servicio de Microbiología-INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña, Spain german.bou.arevalo@sergas.es.
authors.forename: Germán
authors.initials: G
authors.lastname: Bou
mesh_terms: D000667:Ampicillin / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D019818:Clavulanic Acid / Q000494:pharmacology; D016972:Enterobacter cloacae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D041121:Klebsiella oxytoca / Q000187:drug effects / Q000235:genetics; D008826:Microbial Sensitivity Tests; D010397:Penicillanic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D013407:Sulbactam / Q000494:pharmacology; D000078142:Tazobactam; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D019818:Clavulanic Acid; D000667:Ampicillin; D010397:Penicillanic Acid; C048795:AmpC beta-lactamases; D001618:beta-Lactamases; D013407:Sulbactam; D000078142:Tazobactam