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Antimicrobial Agents and Chemotherapy, June 2006, p. 2030-2037, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.01458-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Model System To Evaluate the Effect of ampD Mutations on AmpC-Mediated ß-Lactam Resistance

Amber J. Schmidtke1,2 and Nancy D. Hanson1,2*

Department of Medical Microbiology and Immunology,1 Center for Research in Anti-Infectives and Biotechnology, Creighton University School of Medicine, Omaha, Nebraska2

Received 10 November 2005/ Returned for modification 3 February 2006/ Accepted 31 March 2006

Mutations within the structural gene of ampD can lead to AmpC overproduction and increases in ß-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.

* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178. Phone: (402) 280-5837. Fax: (402) 280-1875. E-mail: ndhanson{at}creighton.edu.

Antimicrobial Agents and Chemotherapy, June 2006, p. 2030-2037, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.01458-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Jacoby, G. A. (2009). AmpC {beta}-Lactamases. Clin. Microbiol. Rev. 22: 161-182 [Abstract] [Full Text]  
  • Schmidtke, A. J., Hanson, N. D. (2008). Role of ampD Homologs in Overproduction of AmpC in Clinical Isolates of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 52: 3922-3927 [Abstract] [Full Text]  
  • Wolter, D. J., Schmidtke, A. J., Hanson, N. D., Lister, P. D. (2007). Increased Expression of ampC in Pseudomonas aeruginosa Mutants Selected with Ciprofloxacin. Antimicrob. Agents Chemother. 51: 2997-3000 [Abstract] [Full Text]  


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