Using groEL as the target for identification of Enterococcus faecium clades and 7 clinically relevant Enterococcus species

Background/Purpose: Accurate identification is important for effective treatment because Enterococcus species have talents to cope with various antibiotics either by intrinsic resistance or by acquisition of mobile genetic elements. The groEL gene is a permissive target in identification of bacteria. We aimed to develop simple assays based on groEL for identification of enterococci. Results: We continued our previous work and determined groEL gene sequences of Enterococcus species isolated from clinical specimens. Phylogenetic analysis based on groEL revealed that each strain clustered well with their reference strains (bootstrap value 100%), in which Enterococcus faecium and Enterococcus gallinarum could be split into two clades. The divergence of E. faecium was coincident with hospital-associated clade, known as clade A, and community-associated clade, known as clade B. A PCR-restriction fragment length polymorphism (PCR-RFLP) assay was therefore designed to differentiate the two E. faecium clades, based on the specific RsaI cutting sites present in the two clades. To differentiate 7 clinical relevant Enterococcus species, the multiplex PCR assay was designed to identify Enterococcus avium, Enterococcus casseliflavus, Enterococcus faecalis, E. faecium, E. gallinarum, Enterococcus hirae and Enterococcus raffinosus. Specificity was tested with other Enterococcus species including Enterococcus cecorum, Enterococcus durans and Enterococcus mundtii. None of these bacterial species generated products of similar size to those of the seven Enterococcus species. Conclusion: The simple PCR-RFLP and multiplex PCR assays on the basis of groEL gene provided an alternative way to identify Enterococcus species. Keywords: Enterococcus, PCR-RFLP, Multiplex PCR, groEL