title: Prevalence and molecular characterization of clinical isolates of Escherichia coli expressing an AmpC phenotype.
abstract: OBJECTIVE
  To establish the prevalence of the AmpC beta-lactamase phenotype in clinical isolates of Escherichia coli and characterize the genetic resistance mechanisms causing the observed phenotype.
  
  
  METHODS
  Clinical E. coli (n = 74) with reduced susceptibility to third-generation cephalosporins and resistance to cefoxitin were collected from the Department of Clinical Microbiology at Hvidovre Hospital, Denmark, in 2006. The AmpC disc test was used to confirm expression of AmpC, and test-positive strains were selected for further antimicrobial susceptibility testing and molecular characterization. Hyperproduction of AmpC beta-lactamase was confirmed by isoelectric focusing (IEF). The presence of a plasmid-mediated ampC gene (pAmpC) was detected by multiplex PCR. The promoter and the entire reading frame of the chromosomal ampC gene were sequenced to identify promoter mutations associated with hyperproduction and gene mutations associated with extended-spectrum AmpC (ESAC) beta-lactamase activity.
  
  
  RESULTS
  Twenty-four isolates exhibited a positive AmpC disc test. IEF confirmed AmpC expression in all isolates except one. Four isolates contained a bla(CMY-2) gene. These were not clonally related by multilocus sequence typing (MLST). The remaining isolates all had mutations or insertions in the promoter region, which could explain increased expression of the chromosomal AmpC enzyme. Mutations in the ampC gene associated with extended activity were rare and did not cause resistance to cefepime. Sequencing of ampC showed that most isolates were not clonally related.
  
  
  CONCLUSIONS
  E. coli expressing an AmpC phenotype occur sporadically and cause significant resistance to cephalosporins. The majority of these are hyperproducing chromosomal ampC although some isolates have acquired pAmpC.
authors.affiliation: Department of Clinical Microbiology 445, Hvidovre Hospital, 2650 Hvidovre, Denmark.
authors.forename: Rikke Lind
authors.initials: RL
authors.lastname: Jørgensen
authors.forename: Jesper Boye
authors.initials: JB
authors.lastname: Nielsen
authors.forename: Alice
authors.initials: A
authors.lastname: Friis-Møller
authors.forename: Hans
authors.initials: H
authors.lastname: Fjeldsøe-Nielsen
authors.forename: Kristian
authors.initials: K
authors.lastname: Schønning
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D003718:Denmark / Q000453:epidemiology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000453:epidemiology* / Q000382:microbiology; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007526:Isoelectric Point; D010641:Phenotype; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015995:Prevalence; D017422:Sequence Analysis, DNA; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D047090:beta-Lactams / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D047090:beta-Lactams; C048795:AmpC beta-lactamases; D001618:beta-Lactamases