Analysis of AmpC β-lactamase expression and sequence in biochemically atypical ceftazidime-resistant Enterobacteriaceae from paediatric patients

Objectives: 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.