Species identification of enterococci via intergenic ribosomal PCR

Accurate species identification of enterococci has become important with the wide prevalence of acquired vancomycin resistance and the presence of less epidemiologically important, inherently vancomycin-resistant enterococci. Using a collection of enterococcal strains, we found that PCR amplification of the intergenic spacer (ITS-PCR) between the 16S and 23S rRNA genes can produce amplicon profiles characteristic of the enterococcus examined. The species examined were group I enterococci (Enterococcus avium, Enterococcus raffinosus, Enterococcus malodoratus, and Enterococcus pseudoavium), group II enterococci (Enterococcus faecalis, Enterococcus faecium, Enterococcus casseliflavus, Enterococcus mundtii, and Enterococcus gallinarum), and group III enterococci (Enterococcus durans and Enterococcus hirae). The enterococcal species in group I, as well as E. faecalis and two strains of E. hirae, were similar and therefore had to be differentiated from each other by Sau3A restriction digests. This produced patterns characteristic of each of these species. The remaining group II and group III enterococcal species produced amplicons characteristic of a particular species except E. gallinarum. The PCR products from E. gallinarum displayed strain-to-strain heterogeneity in the number and size of amplicons. To further test the utility of this technique, 11 phenotypically aberrant strains which had been assigned species identification based on Facklam and Collins-type strain reactions (R. R. Facklam and M. D. Collins, J. Clin. Microbiol. 27:731-734, 1989) were subjected to ITS-PCR. ITS-PCR of the phenotypically aberrant strains identified six strains with reactions consistent with those of type strains. However, five strains were characterized as follows: two strains originally identified as E. mundtii were identified by ITS-PCR as E. casseliflavus, one strain originally identified by ITS-PCR as E. raffinosus was identified as E. durans, one strain originally identified by ITS-PCR as E. hirae was identified as E. faecium, and one strain originally identified as E. durans was identified by ITS-PCR as E. hirae. We conclude that amplification of the intergenic 23S and 16S rRNA gene regions of enterococci provides a reliable technique for species identification of enterococci.