title: Genetic characterization of an extended-spectrum AmpC cephalosporinase with hydrolysing activity against fourth-generation cephalosporins in a clinical isolate of Enterobacter aerogenes selected in vivo.
abstract: BACKGROUND
  Extended-spectrum AmpC cephalosporinases (ESACs) have been reported in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. Here, we characterize a new AmpC variant presenting a broadened substrate activity towards fourth-generation cephalosporins, selected in vivo following cefepime treatment for Enterobacter aerogenes.
  
  
  METHODS
  Two consecutive clonally related isolates of E. aerogenes were evaluated. Screening for ESAC production was performed using plates containing 200 mg/L cloxacillin. MICs were determined by microdilution (CLSI guidelines). bla(AmpC) genes were cloned into a pCR-Blunt II-TOPO vector and expressed in Escherichia coli. The ampC genes were cloned into vector pGEX-6P-1 for protein purification.
  
  
  RESULTS
  Isolate Ea595 was resistant to two fourth-generation cephalosporins, cefepime and cefpirome; using plates containing cloxacillin, susceptibility to ceftazidime and cefepime was restored, suggesting overproduction of the ESAC β-lactamase. Sequencing identified a new AmpC β-lactamase variant presenting one amino acid substitution, Val291Gly, inside the H-10 helix. Recombinant plasmids harbouring this ESAC β-lactamase conferred a broadened resistance profile to cefepime and cefpirome, with resistance levels increasing from 16- to 32-fold in E. coli. AmpC-Ea595 hydrolysed ceftazidime, cefepime and cefpirome at high levels, presenting a lower K(m) and enabling us to classify the enzyme as an ESAC. Homology modelling suggested that the size of the active site could have increased.
  
  
  CONCLUSIONS
  We characterized an ESAC β-lactamase selected in vivo and conferring a high level of resistance to fourth-generation cephalosporins in E. aerogenes. The broadened spectrum was caused by a new modification to the H-10 helix, which modified the active site.
authors: Department of Microbiology, University of Seville, Seville, Spain. jmrodriguez@us.es
authors: Jose M
authors: JM
authors: Rodríguez-Martínez
authors: Pedro
authors: P
authors: Fernández-Echauri
authors: Felipe
authors: F
authors: Fernández-Cuenca
authors: Paula
authors: P
authors: Diaz de Alba
authors: Alejandra
authors: A
authors: Briales
authors: Alvaro
authors: A
authors: Pascual
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism*; D000077723:Cefepime; D002510:Cephalosporinase / Q000235:genetics* / Q000378:metabolism; D002511:Cephalosporins / Q000008:administration & dosage / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D021902:Enterobacter aerogenes / Q000187:drug effects* / Q000302:isolation & purification; D004756:Enterobacteriaceae Infections / Q000188:drug therapy* / Q000382:microbiology; D006868:Hydrolysis; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA
chemical_list: D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D004269:DNA, Bacterial; D000077723:Cefepime; D002510:Cephalosporinase