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Updated: Jan 26, 2026

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance
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Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Published on: May 2, 2018

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Novel trimethoprim resistance gene, dfrA35, in IncC plasmids from Australia

Stephanie J Ambrose1, Ruth M Hall1

  • 1School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales, Australia.

The Journal of Antimicrobial Chemotherapy
|April 17, 2019
PubMed
Abstract

Insights

A novel gene, dfrA35, found on IncC plasmids in Australia, confers trimethoprim resistance in Gram-negative bacteria. This highlights the need for direct testing to identify new resistance mechanisms.

Area of Science:

  • Microbiology
  • Genetics
  • Molecular Biology

Background:

  • Over 30 genes encoding trimethoprim-insensitive dihydrofolate reductase are known in Gram-negative bacteria.
  • These enzymes confer resistance to the antibiotic trimethoprim.

Purpose of the Study:

  • To investigate a putative dihydrofolate reductase gene on type 2 IncC plasmids from Melbourne, Australia (2002-2013).
  • To determine if this gene confers trimethoprim resistance in Gram-negative bacteria.

Main Methods:

  • Plasmid transfer via conjugation to laboratory Escherichia coli.
  • PCR amplification, Gibson Assembly cloning into pUC19, and transformation into E. coli.
  • Broth microdilution for trimethoprim resistance determination.
  • Phylogenetic analysis of the dihydrofolate reductase protein using MEGA and Geneious Prime.

Main Results:

  • The IncC plasmid pEc158 from a 2002 E. coli isolate successfully transferred trimethoprim resistance.
  • A novel dfrA gene, named dfrA35, was identified, encoding a DfrA protein with <40% amino acid identity to known proteins.
  • Cloning and testing confirmed that dfrA35 confers trimethoprim resistance.
  • The dfrA35 gene was located within a complex resistance island on the plasmid, also found in related Klebsiella pneumoniae plasmids.

Conclusions:

  • Novel trimethoprim resistance genes like dfrA35 are continuously discovered.
  • Relying solely on genomic databases for phenotype prediction can lead to missed resistance mechanisms.
  • Direct phenotypic testing remains crucial for accurate identification of antimicrobial resistance determinants.

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