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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.

Development of a PCR Assay for Rapid Detection of Enterococci

Danbing Ke,1,2 François J. Picard,1 Francis Martineau,1,2 Christian Ménard,1 Paul H. Roy,1,3 Marc Ouellette,1,2 and Michel G. Bergeron1,2,*

Centre de Recherche en Infectiologie de l'Université Laval, Sainte-Foy, Québec, Canada G1V 4G2,1 and Division de Microbiologie, Faculté de Medicine,2 and Département de Biochimie, Faculté des Sciences et de Génie,3 Université Laval, Sainte-Foy, Québec, Canada G1K 7P4

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.

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.


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