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

Abstract

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.

Toxin–antitoxin systems are ubiquitous and plasmid-encoded in vancomycin-resistant enterococci

“…PCR with primers complementary to vanA and vanB revealed that 75 of 75 of these strains had plasmids with the vanA gene cluster, and 7 of 75 also possessed plasmids harboring the vanB genes. Through strain typing using a PCR-based species identification method (15)(16)(17)(18)(19), it was determined that eight of the strains were Enterococcus faecalis, and 61 were E. faecium; six strains were not able to be typed by this method and are denoted as Enterococcus spp. A complete list of all strains and the features of their plasmids is in supporting information (SI) Tables 1-3.…”

Isolation and identification of Enterococcus faecalis from necrotic root canals using multiplex PCR

“…One previous study of 22 strains of Enterococcus isolated from the canals of root-filled teeth found no species other than E. faecalis (26). In fact, E. faecalis is reported to account for 80% of enterococcus infections in humans, whereas E. faecium accounts for the remaining 20% (16). Therefore, any false positive identification of E. faecalis in samples obtained from root canals seems unlikely.…”

“…1 and 2, the smaller fragments (650 and 803 bp) correspond to the coding regions of GroES/EL chaperone protein and iron-sulfur binding protein, respectively. The specificity of the chaperone gene was previously demonstrated by testing multiple Enterococcus species (16). The iron-sulfur binding gene was selected randomly in this study.…”

“…On the basis of previous reports (14,16) and genetic databases, three pairs of primers were designed for identification and detection of E. faecalis (Table 1). All the primers were designed according to the full-length sequence of E. faecalis V583, and were synthesized by MWG, Germany.…”

“…PCR-based diagnosis of E. faecalis has been reported by many workers (14)(15)(16)(17)(18)(19)(20)(21).…”