The accurate identification and quantification of six Enterococcus species using quantitative polymerase chain reaction based novel DNA markers

Abstract

Because of their beneficial effects, some enterococci species are used in probiotics, whereas some species are considered opportunistic pathogens implicated in human infections. High 16S rRNA sequence similarities among Enterococcus can impede their accurate classification. In this study, using pan-genome analysis, we developed an accurate method for the identification and quantification of six major Enterococcus species in different foods. Bioinformatics analysis revealed that some Enterococcus faecium genomes deposited in GenBank were misclassified. Six novel markers (FBPase_2 for Enterococcus durans, EcsB for Enterococcus faecalis, PurR for E. faecium, SDF for Enterococcus hirae, HTH_ARSR for Enterococcus lactis, and wxL for Enterococcus mundtii) were mined based on pan-genome analyses and used for quantitative polymerase chain reaction (qPCR). For all species, the detection limits in pure cultures and spiked food samples were 102 colony-forming units/ml (CFU/ml). This method facilitated the sensitive identification of six species in foods and the presence of nontarget bacteria. Moreover, qPCR efficiency was identified using 140 isolates and showed the same or higher resolution when compared with 16S rRNA sequencing. Our qPCR assay will help identify and quantify Enterococcus species in foods, thereby enabling a better understanding of ecological and safety aspects in food samples.