Species Identification of Enterococci via Intergenic Ribosomal PCR (original) (raw)

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 entero-coccus examined. The species examined were group I enterococci (Enterococcus avium, Enterococcus raffinosus, Enterococcus malodoratus, and Enterococcus pseudoavium), group II enterococci (Enterococcus faecalis, Entero-coccus 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. Enterococci are recognized as important agents of nosoco-mially acquired infections such as bacteremia (17). These infections are often difficult to treat due to the increased antibiotic resistance associated with this organism, leaving few therapeutic options (13). As an aid in the management of patients with such infections, species identification can play an important role. For example, determining if two enterococcal isolates recovered from two consecutive blood cultures are the same enterococcal species can be important in the selection of appropriate antibiotics for the treatment of a patient (13, 16). Enterococcal species identification can be a useful tool for epidemiologic surveillance. Species identification of entero-cocci can determine whether a possible outbreak of vancomy-cin-resistant enterococci (VRE) is caused by a species such as Enterococcus faecium, is due to an intrinsically vancomycin-resistant species such as Enterococcus gallinarum, or is caused by an unusual enterococcal species not previously recognized as being vancomycin resistant. Studies have shown that Enterococcus faecalis accounts for 80 to 90% of infections, followed by E. faecium, which accounts for 5 to 10% of infections, and the other enterococcal species, which account for the remainder (6, 13). Species identification of enterococci is usually determined phenotypically by using characteristics such as motility, pigment production, and carbohydrate utilization (6, 18). However, this becomes difficult when unusual strains with few characteristic reactions arise and when nonmotile E. gallinarum or Enterococcus casseliflavus strains are encountered. Dutka-Malen et al. (5) have added the technique of PCR for rapid species identification of E. faecalis, E. faecium, E. casseliflavus, and E. gallinarum based on amplification of the species-specific ligase gene or portion of this gene in these species. However, this technique only identifies the four species mentioned above, leaving the other entero-coccal species unidentifiable except by phenotypic means. Therefore, an alternative rapid DNA-based methodology would be an asset to the identification of the remaining en-terococci to the species level. To explore the use of molecular biology for bacterial species identification, previous investigators have used 16S and 23S rRNA sequences as targets for DNA probes and/or sequencing of these genes (2-4, 9, 19). However, due to evolutionary constraints, there is minimal variation in the 16S or 23S rRNA sequences between species of bacteria. Barry et al. (2) and Jensen et al. (8) overcame the problem of minimal variability by examining the 16S-23S rDNA (genes coding for rRNA) intergenic region, referred to as the internally transcribed spacer region (ITS). It was suggested that this segment of DNA would be under minimal selective pressure compared to the selective pressure that rRNA genes would be under and therefore may allow for species identification due to enhanced variability between species within a genus. This has met with some success for bacterial genera such as Listeria, Escherichia, En-terobacter, and Citrobacter (8). We have further expanded that work to include the genus Enterococcus.