Identification of enterococcal plasmids by multiplex-PCR-based relaxase typing
Abstract number: P1669
Goicoechea P., Romo M., Coque T.M., Baquero F., de la Cruz F., Martínez-Martínez L., Francia M.V.
Objectives: Identification of plasmids is important in order to analyse their distribution and relationships to host cells, their evolutionary origins, and the horizontal gene transfer, a process with a great impact in the spread of antimicrobial resistance. Although methods for the identification of plasmids from enterobacteria were soon developed, no such methods are currently available for enterococcal plasmids. We have developed a novel PCR-based typing method to describe the ecology of the antibiotic resistance plasmids in Enterococcus and evaluate their role in the spread of antimicrobial resistance. Since plasmid transfer is an almost universal procedure for gene spread among bacteria, a typing method based in the relaxase, the crucial protein involved in initiation of transfer, could be widely applicable.
Methods: 14 pairs of primers were designed on the basis of published enterococcal relaxases sequences. 27 reference enterococcal plasmids/strains were first tested using simplex-PCR with each pair of primers for initial testing of the oligonucleotides. 3 Multiplex-PCRs were then performed on the reference plasmids/strains. Sequencing of the amplicons was done by standard procedures for confirmation of the results.
Results: The specificity of each primer pair was tested on two plasmid-free Enterococcus strains: E. faecium BM4105RF and E. faecalis UV202. None of the primer pairs gave any amplification using both templates. Multiplex 1, 2 and 3 correctly detected the relaxases encoded on the reference plasmids and no false positive results were observed.
Conclusions: The method developed is specific and sensitive and does not require a laborious work. Individual or multiple plasmids present in the same cell can be easily detected. Therefore, this approach is potentially applicable for the typing of known enterococcal plasmids present in diverse collections of strains. It will facilitate the understanding of the tremendous spread of the antibiotic resistance in Enterococcus.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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