PCR assay for species identification and virulence genes among clinical isolates of Enterococcus

Identification of Enterococcus to the species level by conventional methods is time-consuming and complicated. Accurate identification of bacteria is important for effective treatment. In this study, we developed a multiplex PCR assay targeting groEL gene to identify Enterococcus to the species level. Species-specific primers from 9 species were designed based on the variable regions of each groEL gene. Each species generated different size (141 to 745 bp) except E. avium/ E. raffinosus and E. hirae/ E. mundtii. Further digestion of PCR products with Hind III can distinguish E. avium from E. raffinosus and E. hirae from E. mundtii. A total of 114 clinical isolates were tested and showed that the assay had 100% agreement with identification by 16S rDNA sequence. This multiplex PCR assay is easy to perform and accurate for identification of Enterococcus species including E. faecalis/ faecium and non- E. faecalis/ faecium species. In another part, three pairs of primers were combined to differentiate common gram positive cocci, including Staphylococcus aureus, Streptococcus species, and Enterococcus species.
Furthermore, the prevalence of esp and hyl of E. faecium was also investigated. E. faecium is generally considered to be a species of limited virulence. Recently, some data suggest that E. faecium strains may have become more and more virulent. esp and hyl are potential virulence genes of E. faecium strains. Besides, in our previous study, we found that E. faecium clinical isolates could group into two PCR-RFLP types based on groESL genes. The aim of the study was to find the correlation between different PCR-RFLP types of E. faecium and esp, hyl virulence genes. Of 86 strains tested, both determinants were found predominately in groESL PCR-RFLP type I strains. All PCR-RFLP type II strains have neither esp nor hyl virulence genes. However, comparing VRE with non-VRE, both esp and hyl virulence genes were mainly present in VRE.