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A classification system for plasmids from enterococci and other Gram-positive bacteria.

L B Jensen1, L Garcia-Migura, A J S Valenzuela

  • 1National Food Institute, DTU, Division of Microbiology and Risk Assessment, Bülowsvej 27, DK-1790 Copenhagen V, Denmark. lboj@food.dtu.dk

Journal of Microbiological Methods
|November 3, 2009
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Summary

A new plasmid classification system for Gram-positive bacteria, focusing on enterococci, identifies 19 replication initiator gene families. This system reveals distinct prevalence patterns in Enterococcus faecalis and Enterococcus faecium, aiding in understanding plasmid diversity.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Plasmids are crucial mobile genetic elements in bacteria, often carrying antibiotic resistance genes.
  • A standardized classification system for plasmids in Gram-positive bacteria, particularly enterococci, is needed for better epidemiological tracking and understanding of genetic exchange.
  • Previous classification efforts have been limited in scope or applicability to diverse Gram-positive species.

Purpose of the Study:

  • To develop and validate a comprehensive classification system for plasmids from enterococci and other Gram-positive bacteria.
  • To determine the prevalence of defined plasmid replicon families in clinical and animal isolates of Enterococcus faecalis and Enterococcus faecium.
  • To investigate the presence of novel or uncharacterized plasmid replicons in enterococci through conjugation experiments.

Main Methods:

  • A classification system was developed by analyzing 111 published plasmid sequences from enterococci and staphylococci.
  • Polymerase Chain Reaction (PCR) amplification of conserved replication initiator (rep) gene regions was used.
  • Sequence alignment and a cutoff value of 80% identity at both protein and DNA levels defined 19 replicon families (rep-families).
  • The prevalence of these rep-families was tested on 79 enterococcal isolates from human and animal sources.
  • Conjugation experiments were performed to identify plasmids in transconjugants lacking the tested rep-families.

Main Results:

  • Nineteen distinct rep-families were defined, along with several unique sequences, based on conserved rep gene regions.
  • Significant differences in rep-family prevalence were observed between Enterococcus faecalis (rep(9) most prevalent) and Enterococcus faecium (rep(2) most prevalent).
  • A substantial proportion of isolates (33% of E. faecium, 32% of E. faecalis) yielded no positive amplicons for the tested rep-families.
  • Conjugation experiments identified 30 transconjugants, 19 of which carried plasmids with rep-families not included in the initial classification system.

Conclusions:

  • The developed classification system provides a framework for categorizing plasmids in Gram-positive bacteria, particularly enterococci.
  • The study highlights species-specific prevalence patterns of plasmid replicons within enterococci.
  • A significant number of enterococcal plasmids harbor uncharacterized or novel replicon types, underscoring the need for ongoing surveillance and classification efforts.