JAC Advance Access originally published online on April 14, 2003
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Journal of Antimicrobial Chemotherapy (2003) 51, 1141-1151
© 2003 The British Society for Antimicrobial Chemotherapy
Factors influencing gene expression and resistance for Gram-negative organisms expressing plasmid-encoded ampC genes of Enterobacter origin
1 Department of Medical Microbiology and Immunology; 2 Center for Research in Anti-Infectives and Biotechnology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA
Received 5 August 2002; returned 13 December 2002; revised 11 February 2003; accepted 14 February 2003
High-level expression of AmpC ß-lactamases results in organisms resistant to multiple ß-lactam antibiotics. The mechanism of chromosomally mediated AmpC resistance has been elucidated, however the mechanism(s) driving plasmid-encoded AmpC resistance are unknown. Studies were designed to identify factors which influence expression of plasmid-encoded ampC genes and correlate these factors with resistance. As the model system, ampC genes of Enterobacter origin were used to determine how gene copy number, genetic background and genetic organization influenced resistance phenotypes. To this end, gene expression from the plasmid-encoded inducible blaACT-1 and non-inducible blaMIR-1 were compared with chromosomal ampC gene expression from both wild-type (WT) and derepressed Enterobacter cloacae isolates. RNA levels within the original clinical isolates were examined using primer extension analysis, whereas a new PCR strategy was developed to examine gene copy number. These data revealed that blaACT-1 and blaMIR-1 constitutive expression was 33- and 95-fold higher than WT expression, whereas copy numbers of the plasmid-encoded genes were 2 and 12, respectively. Differences in promoters and transcriptional starts for the respective plasmid-encoded genes were noted and contribute to increases observed in overall expression. Finally, ß-lactam MICs were increased two- to 16-fold when blaACT-1 was expressed in Escherichia coli AmpD– strains compared with E. coli AmpD+ strains. In conclusion, high-level expression of plasmid-encoded ampC genes requires interplay between multiple factors including genetic organization, promoter modifications, genetic background, and to some extent gene copy number. In addition, clinical laboratories need to be aware that genetic backgrounds of inducible plasmid-encoded genes can dramatically influence MICs for organisms not normally associated with derepressed phenotypes.
Keywords: AmpC expression, resistance, plasmid-encoded, derepressed, copy number
* Corresponding author. Tel: +1-402-280-5837; Fax: +1-402-280-1875; E-mail: ndhanson{at}creighton.edu
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