Development of a PCR assay for rapid detection of enterococci

Enterococci are becoming major nosocomial pathogens, and increasing resista nce to vancomycin has been well documented. Conventional identification met hods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enteroco cci in a variety of clinical specimens and is capable of yielding results i n just a few hours. However, all PCR-based assays developed so far are spec ies specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus lev el by targeting the tuf gene, which encodes elongation factor EF-Tu. Initia lly, we compared the nucleotide sequences of the tuf gene from several bact erial species (available in public databases) and designed degenerate PCR p rimers 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 n ucleotide sequences of these amplicons were determined. The analysis of a m ultiple alignment of these sequences revealed regions conserved among enter ococci but distinct from those of other bacteria. PCR primers complementary to these regions allowed amplification of genomic DNAs from 14 of 15 speci es of enterococci tested (E. solitarius DNA could not be amplified). There was no amplification with a majority of 79 nonenterococcal bacterial specie s, except for 2 Abiotrophia species and several Listeria species. Furthermo re, this assay efficiently amplified all 159 clinical isolates of enterococ ci tested (61 E. faecium, 77 E. faecalis, 9 E. gallinarum, and 12 E. cassel iflavus isolates). Interestingly, the preliminary sequence comparison of th e amplicons for four enterococcal species demonstrated that there were some sequence variations which may be used to generate species-specific interna l 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.