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

Abstract

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...

Prevalence and Antibiotic-Resistance Characteristics of Enterococcus Spp. Isolated From Free-Living and Captive Raptors in Central Illinois

“…All susceptibility test methods were performed as recommended by the manufac- All isolates were confirmed to be Enterococcus spp. by polymerase chain reaction (PCR) with the use of the primers listed in Table 1 (minor modifications of those published by Ke et al, 1999;Dutka-Malen et al, 1995;Angeletti et al, 2001). Bacterial DNA was purified with the use of the QiAmpH kit (Qiagen, Inc., Valencia, California, USA) protocol for bacterial cultures.…”

Application of multilocus sequence analysis (MLSA) for rapid identification of Enterococcus species based on rpoA and pheS genes

“…16S rRNA gene sequencing, DNA-DNA hybridization and SDS-PAGE of whole-cell proteins are among the most common techniques currently used for Enterococcus species identification (Angeletti et al, 2001;Domig et al, 2003;Vancanneyt et al, 2001). However, 16S rRNA gene sequences have limited discriminating power for several closely related enterococcal species, e.g.…”

Phylogeny and Identification of Enterococci by atpA Gene Sequence Analysis

“…Several molecular biology-based techniques, such as multilocus sequence typing, randomly amplified polymorphic DNA (RAPD) analysis, 16S rRNA gene sequencing, amplified fragment length polymorphism (AFLP) analysis, pulsed-field gel electrophoresis (PFGE), and intergenic ribosomal PCR, have been used to identify enterococci to the species and the strain levels (1,2,3,5,8,17,26). AFLP analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) are among the most reliable techniques currently used for Enterococcus species identification (39).…”