Division of the genus Enterococcus into species groups using PCR-based molecular typing methods

Monstein, Hans‐Jürg; Quednau, M.; Samuelsson, Annika; Ahrné, Siv; Isaksson, Barbro; Jonasson, Jon

doi:10.1099/00221287-144-5-1171

Cited by 52 publications

(38 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The application of small-subunit (16S) ribosomal rRNA gene sequencing to the identification of aerobic catalase-negative gram-positive cocci was recently evaluated by Bosshard et al (3). Besides 16S rRNA genes, a variety of other target genes have been used for the identification of bacterial pathogens; for example, the tRNA intergenic spacer, ddl, sod, or tuf gene has been used for the identification of Enterococcus species by many researchers (1,2,6,16,20,24,35,36). Previously, we determined the full-length sequence of the E. faecalis groESL genes containing groES (282 bp), spacer, and groEL (1,623 bp) and developed a species identification method for E. faecalis based on this sequence instead of 16S rRNA gene sequences (31).…”

mentioning

confidence: 99%

Identification of Clinically Relevant Enterococcus Species by Direct Sequencing of groES and Spacer Region

Tsai¹,

Hsueh

Lin

et al. 2005

J Clin Microbiol

View full text Add to dashboard Cite

We determined the groESL sequences (groES, groEL, and the intergenic spacer) of 10 clinically relevant Enterococcus species and evaluated the feasibility of identifying Enterococcus species on the basis of these sequences. Seven common clinical Enterococcus species, E. faecalis, E. faecium, E. casseliflavus, E. gallinarum, E. avium, E. raffinosus, and E. hirae, and three less common Enterococcus species, E. cecorum, E. durans, and E. mundtii, were examined in this study. We found that the groES genes of these enterococcal species are identical in length (285 nucleotides) and contain an unusual putative start codon, GTG. The lengths and sequences of the intergenic regions (spacers between the groES and groEL genes) are quite variable (17 to 57 bp in length) among Enterococcus species but are conserved in strains within each species, with only a few exceptions. Considerable variation of groES or groEL sequences was also observed. The evolutionary trees of groES or groEL sequences revealed similarities among Enterococcus species. However, the overall intraspecies variation of groES was less than that of groEL. The high interspecies variation and low intraspecies variation indicate that the groES and spacer sequences are more useful than groEL for identification of clinically relevant Enterococcus species. The sequences of these two genetic traits, groES and spacer, can be determined by a single PCR and direct sequencing and may provide important information for the differentiation of closely related species of Enterococcus.

show abstract

mentioning

confidence: 99%

Identification of Clinically Relevant Enterococcus Species by Direct Sequencing of groES and Spacer Region

Tsai¹,

Hsueh

Lin

et al. 2005

J Clin Microbiol

View full text Add to dashboard Cite

show abstract

“…It has been succesfully used to identify vancomycin-resistant enterococci (8,13,18,21,22,25,26,30,31), but it may also be used broadly to identify all enterococci; nevertheless, this approach certainly fails to identify some enterococcal species outside the reach of the primers, and for this reason it is also necessary to include among the proposed molecular methods for enterococcal species identification the amplification and sequencing of the 16S ribosomal DNA (rDNA) gene (25,28,38). Two hundred seventy-nine clinical strains consecutively identified as enterococci by three hospital laboratories in Rome were studied ( Table 1 All strains submitted as glycopeptide resistant were verified with the E-test method (AB BIODISK, Solna, Sweden) in our laboratory.…”

mentioning

confidence: 99%

Routine Molecular Identification of Enterococci by Gene-Specific PCR and 16S Ribosomal DNA Sequencing

et al. 2001

View full text Add to dashboard Cite

For 279 clinically isolated specimens identified by commercial kits as enterococci, genotypic identification was performed by two multiplex PCRs, one with ddl E. faecalis and ddl E. faecium primers and another with vanC-1 and vanC-2/3 primers, and by 16S ribosomal DNA (rDNA) sequencing. For 253 strains, phenotypic and genotypic results were the same. Multiplex PCR allowed for the identification of 13 discordant results. Six strains were not enterococci and were identified by 16S rDNA sequencing. For 5 discordant and 10 concordant enterococcal strains, 16S rDNA sequencing was needed. Because many supplementary tests are frequently necessary for phenotypic identification, the molecular approach is a good alternative.Identification at the species level of enterococci isolated from clinical specimens is considered necessary, as is quantitative evaluation of their resistance to penicillin, ampicillin, vancomycin, and teicoplanin and high-level resistance to gentamicin and streptomycin (11). It is also necessary to distinguish the low-virulence motile enterococcal species with constitutive low-level resistance to vancomycin from the species that are more frequently isolated from clinical specimens, such as Enterococcus faecalis and E. faecium, which in some countries can often show high-level inducible and transmissible resistance to glycopeptides (37).Commercially available kits are often used by clinical laboratories as an alternative to the numerous physiological tests needed to identify enterococcal species (6, 9, 10, 11, 37); nevertheless, all commercial kits vary in their performance and persistently show many drawbacks, especially in cases of atypical strains, and at best need supplementary manual tests, which somewhat impair their usefulness (14, 17, 23, 33, 34, 36; P. A. d'Azevedo, C. G. Dias, A. L. S. Gonçalves, F. Rowe, and L. M. Teixeira, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. C-242, 2000). In some cases identification of atypical E. faecium strains may be problematic, as is distinguishing E. gallinarum from E. faecium and also identifying E. durans, E. avium, E. raffinosus, E. hirae, and E. mundtii (1, 2, 6, 7, 35, 37).In 1995 a multiplex PCR was devised for the identification of E. faecium and E. faecalis by primers targeted at specific sequences in the ddl (D-Ala-D-Ala ligase) chromosomal genes of the two species and in the glycopeptide resistance ligase genes vanA, vanB, and vanC (8). The vanC gene is present in the motile, low-level constitutive glycopeptide-resistant species E. gallinarum (vanC-1) and E. casseliflavus and E. flavescens (vanC-2/3); thus, demonstration of its presence indicates the presence of one of the aforementioned species (4,24,27,29,30).The use of a PCR with primers for ddl E. faecalis and ddl E. faecium , together with primers for vanC-1, -2, and -3, may be the most simple molecular approach for both rapid and precise identification of enterococci while avoiding the drawbacks of commercial kits. It has been succesfully used to identify vancomycin-resistant enteroco...

show abstract

“…Another approach to species identification may be the use of molecular methods. Several DNA-based techniques for identifying clinical isolates have been developed (5,6,15,18). A variety of conserved genes, including 16S rRNA genes, the tRNA intergenic spacer, the D-alanine:D-alanine ligase gene (ddl gene), the sodA gene encoding superoxide dismutase, penicillin-binding protein 5, and the elongation factor tuf gene have been used for identification of enterococci (15,20,21,23,30).…”

mentioning

confidence: 99%

Determination of Enterococcus faecalis groESL Full-Length Sequence and Application for Species Identification

et al. 2001

View full text Add to dashboard Cite

Amplification of the partial Cpn60 (or GroEL) gene segment has been used for identification of many bacteria, including Enterococcus species. To obtain more sequence data from groESL genes of Enterococcus faecalis, the full-length sequence of the E. faecalis groESL genes containing groES (285 bp), spacer (57 bp), and groEL (1,626 bp) was determined. A database search of GenBank revealed that the deduced E. faecalis GroES and GroEL proteins show significant homology to the GroES and GroEL proteins of other bacteria. The GroEL (groEL) of E. faecalis had the highest identity with Streptococcus pneumoniae (81.8% amino acid sequence identity and 73.0% nucleotide sequence identity), followed by Lactococcus zeae, while GroES (groES) had 60.2% (64.6%) identity with Lactobacillus zeae and 58.5% (66.2%) identity with Lactococcus lactis, followed by 57.0% (65.5%) identity with Bacillus subtilis. Based on the groES sequence, an E. faecalis-specific PCR assay was developed, and this PCR assay was positive for all the E. faecalis strains tested. Dot blot hybridization using either groES or groEL as the probe distinguished E. faecalis clearly from other species, indicating that both genes can be used as suitable targets for E. faecalis identification. Moreover, broad-range PCR-restriction fragment length polymorphism of groESL was designed to differentiate eight commonly encountered Enterococcus species. The Enterococcus species of reference strains could be easily differentiated on the basis of restriction patterns produced by HaeIII and RsaI. The DNA-based assays developed in this study provide an alternative to currently used methods of identification for clinically important enterococcal species.

show abstract

Division of the genus Enterococcus into species groups using PCR-based molecular typing methods

Cited by 52 publications

References 29 publications

Identification of Clinically Relevant Enterococcus Species by Direct Sequencing of groES and Spacer Region

Identification of Clinically Relevant Enterococcus Species by Direct Sequencing of groES and Spacer Region

Routine Molecular Identification of Enterococci by Gene-Specific PCR and 16S Ribosomal DNA Sequencing

Determination of Enterococcus faecalis groESL Full-Length Sequence and Application for Species Identification

Contact Info

Product

Resources

About