Journal of Clinical Microbiology, November 1999, p. 3497-3503, Vol. 37, No. 11
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Centre de Recherche en Infectiologie de
l'Université Laval, Sainte-Foy, Québec, Canada G1V
4G2,1 and Division de Microbiologie,
Received 22 March 1999/Returned for modification 4 June
1999/Accepted 23 July 1999
Enterococci are becoming major nosocomial pathogens, and increasing
resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus
level by targeting the tuf gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the
tuf gene from several bacterial species (available in
public databases) and designed degenerate PCR primers derived from
conserved regions. These primers were used to amplify a target region
of 803 bp from four enterococcal species (Enterococcus
avium, E. faecalis, E. faecium, and
E. gallinarum). Subsequently, the complete nucleotide
sequences of these amplicons were determined. The analysis of a
multiple alignment of these sequences revealed regions conserved among
enterococci but distinct from those of other bacteria. PCR primers
complementary to these regions allowed amplification of genomic DNAs
from 14 of 15 species of enterococci tested (E. solitarius DNA could not be amplified). There was no amplification with a majority
of 79 nonenterococcal bacterial species, except for 2 Abiotrophia species and several Listeria
species. Furthermore, this assay efficiently amplified all 159 clinical
isolates of enterococci tested (61 E. faecium, 77 E. faecalis, 9 E. gallinarum, and 12 E. casseliflavus isolates). Interestingly, the preliminary sequence
comparison of the amplicons for four enterococcal species demonstrated
that there were some sequence variations which may be used to generate
species-specific internal probes. In conclusion, this rapid PCR-based
assay is capable of detecting all clinically important enterococci and
has potential for use in clinical microbiology laboratories.
*
Corresponding author. Mailing address: Centre de
Recherche en Infectiologie, Centre Hospitalier Universitaire de
Québec, Pavillon CHUL, 2705 Boul. Laurier, Sainte-Foy,
Québec, Canada G1V 4G2. Phone: (418) 654-2705. Fax: (418)
654-2715. E-mail:
Michel.G.Bergeron{at}crchul.ulaval.ca.
This article has been cited by other articles:
| Antimicrob. Agents Chemother. | Clin. Microbiol. Rev. |
|---|---|
| Clin. Vaccine Immunol. | ALL ASM JOURNALS |
|---|