Factors influencing gene expression and resistance for Gram-negative organisms expressing plasmid-encoded ampC genes of Enterobacter origin

Mark Reisbig, Ashfaque Hossain, Nancy D. Hanson

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

High-level expression of AmpC β-lactamases results in organisms resistant to multiple β-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, β-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.

Original languageEnglish
Pages (from-to)1141-1151
Number of pages11
JournalJournal of Antimicrobial Chemotherapy
Volume51
Issue number5
DOIs
StatePublished - May 1 2003

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Enterobacter
Plasmids
Gene Expression
Gene Dosage
Genes
Lactams
Escherichia coli
Phenotype
Enterobacter cloacae
R Factors
Genetic Promoter Regions
RNA
Anti-Bacterial Agents
Polymerase Chain Reaction

All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Microbiology

Cite this

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title = "Factors influencing gene expression and resistance for Gram-negative organisms expressing plasmid-encoded ampC genes of Enterobacter origin",
abstract = "High-level expression of AmpC β-lactamases results in organisms resistant to multiple β-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, β-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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N2 - High-level expression of AmpC β-lactamases results in organisms resistant to multiple β-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, β-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.

AB - High-level expression of AmpC β-lactamases results in organisms resistant to multiple β-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, β-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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