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Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes
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Persistent, toxin-antitoxin system-independent, tetracycline resistance-encoding plasmid from a dairy Enterococcus

Xinhui Li1, Valente Alvarez, Willis James Harper

  • 1Department of Food Science, The Ohio State University, Columbus, OH 43210-1007, USA.

Applied and Environmental Microbiology
|July 26, 2011
PubMed

Insights

A dairy Enterococcus faecium plasmid carrying tetracycline resistance genes (tet(M) and tet(L)) showed high persistence. This plasmid utilizes a toxin-antitoxin-independent mechanism, contributing to antibiotic resistance gene stability.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Antibiotic resistance in bacteria is a growing public health concern.
  • Plasmids play a crucial role in the dissemination of antibiotic resistance genes.
  • Enterococcus faecium is a significant opportunistic pathogen often associated with nosocomial infections.

Purpose of the Study:

  • To investigate the genetic basis and persistence mechanisms of a tetracycline resistance plasmid (pM7M2) in Enterococcus faecium.
  • To determine the transferability and functional expression of the tet(M) and tet(L) genes.
  • To explore the plasmid's stabilization system and its evolutionary relationship with other plasmids.

Main Methods:

  • Plasmid isolation and DNA sequencing.
  • Conjugation experiments for plasmid transfer.
  • Electroporation and natural transformation for gene transfer into other bacterial species.
  • Southern hybridization for gene integration analysis.
  • Quantitative real-time reverse transcription-PCR (RT-PCR) for gene expression analysis.

Main Results:

  • The 19.6-kb plasmid pM7M2 harbored tet(M) and tet(L) genes and demonstrated high stability (99% retention) even without tetracycline pressure.
  • Sequence analysis revealed a plasmid backbone homologous to pRE25, lacking a canonical toxin-antitoxin system but exhibiting TA-independent stabilization.
  • The tet(M)-tet(L) gene cluster was functional, conferring tetracycline resistance upon transfer to Enterococcus faecalis and Streptococcus mutans, with chromosomal integration observed in S. mutans.
  • Tetracycline-induced transcription of both tet(M) and tet(L) genes was confirmed via RT-PCR.

Conclusions:

  • The plasmid pM7M2 employs a TA-independent mechanism contributing to the stable maintenance of antibiotic resistance genes.
  • The tet(M) and tet(L) genes are functional and transferable, potentially increasing the spread of tetracycline resistance.
  • Evolutionary links between pM7M2, pIP816, and pRE25 are suggested, highlighting the dynamic nature of plasmid evolution and antibiotic resistance dissemination.

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