Detection of vancomycin resistance genes combined with typing of Enterococci by means of multiplex PCR amplification and multiple primer DNA sequencing

• A multiplex PCR assay for the detection of vancomycin resistance (van) genes in enterococci was established. Primers targeting the 16S rRNA gene were included in the reaction mixture. Multiple-primer DNA sequencing of the PCR products provided species identification through partial nucleotide sequences of 16S rRNA genes, as well as confirmation of the correct identification of vanA, vanB, vanC-1, and vanC-2/3 genotypes. Thirty-nine enterococcal clinical isolates and type strains were examined for the presence of vancomycin resistance determinants. Twelve other isolates from a clinical reference collection (some of them having vanA, vanB, vanC-1, or vanC-2/3 genotypes) were used as controls. Hybridization and partial DNA sequence analysis of multiplex PCR products revealed that none of the clinical isolates had a vanA genotype and only one had a vanB genotype, vanC- 1 was found in three clinical isolates, and vanC-2/3 in one. Results obtained with the reference and type strains were in agreement with earlier results.

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