title: Mutational events in cefotaximase extended-spectrum beta-lactamases of the CTX-M-1 cluster involved in ceftazidime resistance.
abstract: CTX-M beta-lactamases, which show a high cefotaxime hydrolytic activity, constitute the most prevalent extended-spectrum beta-lactamase (ESBL) type found among clinical isolates. The recent explosive diversification of CTX-M enzymes seems to have taken place due to the appearance of more efficient enzymes which are capable of hydrolyzing both cefotaxime and ceftazidime, especially among the CTX-M-1 cluster. A combined strategy of in vitro stepwise evolution experiments using bla(CTX-M-1), bla(CTX-M-3), and bla(CTX-M-10) genes and site-directed mutagenesis has been used to evaluate the role of ceftazidime and other beta-lactam antibiotics in triggering the diversity found among enzymes belonging to this cluster. Two types of mutants, P167S and D240G, displaying high ceftazidime MICs but reduced resistance to cefotaxime and/or cefepime, respectively, were identified. Such an antagonistic pleiotropic effect was particularly evident with P167S/T mutations. The incompatibility between P167S and D240G changes was demonstrated, since double mutants reduced susceptibility to both ceftazidime and cefotaxime-cefepime; this may explain the absence of strains containing both mutations in the clinical environment. The role of A77V and N106S mutations, which are frequently associated with P167S/T and/or D240G, respectively, in natural strains, was investigated. The presence of A77V and N106S contributes to restore a high-level cefotaxime resistance phenotype, but only when associated with mutations P167S and D240G, respectively. However, A77V mutation increases resistance to both cefotaxime and ceftazidime when associated with CTX-M-10. This suggests that in this context this mutation might be considered a primary site involved in resistance to broad-spectrum cephalosporins.
authors.affiliation: Hospital Universitario Ramón y Cajal, IMSALUD, Madrid, Spain.
authors.forename: Angela
authors.initials: A
authors.lastname: Novais
authors.forename: Rafael
authors.initials: R
authors.lastname: Cantón
authors.forename: Teresa M
authors.initials: TM
authors.lastname: Coque
authors.forename: Andrés
authors.initials: A
authors.lastname: Moya
authors.forename: Fernando
authors.initials: F
authors.lastname: Baquero
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Galán
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001483:Base Sequence; D002439:Cefotaxime / Q000494:pharmacology; D002442:Ceftazidime / Q000494:pharmacology*; D018550:Cephalosporin Resistance / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D019143:Evolution, Molecular; D005798:Genes, Bacterial; D014644:Genetic Variation; D006801:Humans; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004269:DNA, Bacterial; D002442:Ceftazidime; C055184:beta-lactamase TEM-3; D001618:beta-Lactamases; D002439:Cefotaxime