JAC Advance Access published online on April 14, 2003
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkg204
© 2003 by The British Society for Antimicrobial Chemotherapy
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Original article
1 Department of Medical
Microbiology and Immunology; Center
for Research in Anti-Infectives and Biotechnology, Creighton University
School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA
* Corresponding author. E-mail: ndhanson{at}creighton.edu.
Received 5 August 2002
; revised 13 December 2002
; accepted 14 February 2003
High-level expression of AmpC
Keywords: AmpC expression, resistance, plasmid-encoded,
derepressed, copy number
Factors influencing gene expression and resistance
for Gram-negative organisms expressing plasmid-encoded ampC genes
of
Enterobacter origin
-lactamases
results in organisms resistant to multiple -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, -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.
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