Persistent, toxin-antitoxin system-independent, tetracycline resistance-encoding plasmid from a dairy Enterococcus...

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.