Model system to evaluate the effect of ampD mutations on AmpC-mediated β-lactam resistance

Amber J. Schmidtke, Nancy D. Hanson

Research output: Contribution to journalArticle

36 Scopus citations

Abstract

Mutations within the structural gene of ampD can lead to AmpC overproduction and increases in β-lactam MICs in organisms with an inducible ampC. However, identification of mutations alone cannot predict the impact that those mutations have on AmpD function. Therefore, a model system was designed to determine the effect of ampD mutations on ceftazidime MICs using an AmpD- mutant Escherichia coli strain which produced an inducible plasmid-encoded AmpC. ampD genes were amplified by PCR from strains of E. coli, Citrobacter freundii, and Pseudomonas aeruginosa. Also, carboxy-terminal truncations of C. freundii ampD genes were constructed representing deletions of 10, 21, or 25 codons. Amplified ampD products were cloned into pACYC184 containing inducible blaACT-1-ampR. Plasmids were transformed into E. coli strains JRG582 (AmpD-) and K-12 259 (AmpD+). The strains were evaluated for a derepressed phenotype using ceftazidime MICs. Some mutated ampD genes, including the ampD gene of a derepressed C. freundii isolate, resulted in substantial decreases in ceftazidime MICs (from >256 μg/ml to 12 to 24 μg/ml) for the AmpD- strain, indicating no role for these mutations in derepressed phenotypes. However, ampD truncation products and ampD from a partially derepressed P. aeruginosa strain resulted in ceftazidime MICs of >256 μg/ml, indicating a role for these gene modifications in derepressed phenotypes. The use of this model system indicated that alternative mechanisms were involved in the derepressed phenotype observed in strains of C. freundii and P. aeruginosa. The alternative mechanism involved in the derepressed phenotype of the C. freundii isolate was downregulation of ampD transcription.

Original languageEnglish (US)
Pages (from-to)2030-2037
Number of pages8
JournalAntimicrobial agents and chemotherapy
Volume50
Issue number6
DOIs
StatePublished - Jun 1 2006

All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases

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