title: Analysis of AmpC beta-lactamase expression and sequence in biochemically atypical ceftazidime-resistant Enterobacteriaceae from paediatric patients.
abstract: OBJECTIVE
  To analyse the variation of ampC beta-lactamase gene sequence and expression in biochemically atypical Enterobacteriaceae isolates, and to identify them definitively.
  
  
  METHODS
  beta-Lactamase gene-containing recombinant plasmids transformed into Escherichia coli were selected using ampicillin. PCR analysis was used to locate specific ampC and 16S rRNA genes, and the amplicons were sequenced. Random amplified polymorphic DNA PCR was used to group isolates and API 20E biochemical profiling was used to identify them putatively.
  
  
  RESULTS
  Of 50 ceftazidime-resistant clinical Enterobacteriaceae isolates, 36 were identified (>95% confidence)-using API 20E test strips-as being organisms known to express inducible class C beta-lactamases (Citrobacter freundii, Enterobacter cloacae, Morganella morganii or Hafnia alvei). The rest were biochemically atypical. Of these, isolate I113, putatively identified as E. coli, possesses a chromosomally encoded ampC which differs by 15% from C. freundii OS60 ampC and by >30% from E. coli ampC. A related ampC gene was found in another seven of the atypical isolates. The use of various identification methods, including ampC sequence analysis, revealed that these I113-like ampC-positive isolates represent Citrobacter murliniae and Citrobacter youngae.
  
  
  CONCLUSIONS
  We report sequences for two new Citrobacter spp. ampC genes, and provide evidence that ampC sequencing is a discriminatory method for identifying atypical Citrobacter spp. isolates.
authors.affiliation: Bristol Centre for Antimicrobial Research and Evaluation, Department of Pathology & Microbiology, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK. Matthewb.Avison@bris.ac.uk
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Avison
authors.forename: Sarah
authors.initials: S
authors.lastname: Underwood
authors.forename: Aki
authors.initials: A
authors.lastname: Okazaki
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Walsh
authors.forename: Peter M
authors.initials: PM
authors.lastname: Bennett
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D002442:Ceftazidime / Q000494:pharmacology / Q000627:therapeutic use; D018550:Cephalosporin Resistance / Q000235:genetics*; D002648:Child; D004755:Enterobacteriaceae / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics*; D005798:Genes, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D002442:Ceftazidime; C048795:AmpC beta-lactamases; D001618:beta-Lactamases