Distinction between Enterococcus faecium and Enterococcus lactis by a gluP PCR-Based Assay for Accurate Identification and Diagnostics

Abstract

It was recently proposed that Enterococcus faecium colonizing the human gut (previous clade B) actually corresponds to Enterococcus lactis. Our goals were to develop a PCR assay to rapidly differentiate these species and to discuss the main phenotypic and genotypic differences from a clinical perspective. The pan-genome of 512 genomes of E. faecium and E. lactis strains was analyzed to assess diversity in genes between the two species. Sequences were aligned to find the best candidate gene for designing species-specific primers, and their accuracy was tested with a collection of 382 enterococci. E. lactis isolates from clinical origins were further characterized by whole-genome sequencing (Illumina). Pan-genome analysis resulted in 12 gene variants, with gene gluP (rhomboid protease) being selected as the candidate for species differentiation. The nucleotide sequence of gluP diverged by 90 to 92% between sets, which allowed species identification through PCR with 100% specificity and no cross-reactivity. E. lactis strains were greatly pan-susceptible and not host specific. Hospital E. lactis isolates were susceptible to clinically relevant antibiotics, lacked infection-associated virulence markers, and were associated with patients presenting risk factors for enhanced bacterial translocation. Here, we propose a PCR-based assay using gluP for easy routine differentiation between E. faecium and E. lactis that could be implemented in different public health contexts. We further suggest that E. lactis, a dominant human gut species, can cross the gut barrier in severely ill, immunodeficient, and surgical patients. Knowing that bacterial translocation may be a sepsis promoter, the relevance of infections caused by E. lactis strains, even if they are pan-susceptible, should be explored. IMPORTANCE Enterococcus faecium is a WHO priority pathogen that causes severe and hard-to-treat human infections. It was recently proposed that E. faecium colonizing the human gut (previous clade B) actually corresponds to Enterococcus lactis; therefore, some of the human infections occurring globally are being misidentified. In this work, we developed a PCR-based rapid identification method for the differentiation of E. faecium and E. lactis and discussed the main phenotypic and genotypic differences of these species from a clinical perspective. We identified the gluP gene as the best candidate, based on the phylogenomic analysis of 512 published pan-genomes, and validated the PCR assay with a comprehensive collection of 382 enterococci obtained from different sources. Further detailed analysis of clinical E. lactis strains showed that they are highly susceptible to antibiotics and lack the typical virulence markers of E. faecium but are able to cause severe human infections in immunosuppressed patients, possibly in part due to gut barrier translocation.

Keywords: Enterococcus faecium; Enterococcus lactis; PCR-based differentiation; gluP; public health.

FIG 1

(A) Maximum likelihood phylogenetic tree of E. faecium and E. lactis based on core genome alignment, representing a clear clade separation of clade A1 E. faecium (red), clade-A2 E. faecium (blue), and clade B E. faecium/E. lactis (green). (B) Presence and absence matrix of the core and accessory genomes with respect to their phylogenetic positions. (C) Alignment of the gluP gene, showing the two main gene sequences from clade A/E. faecium (GenBank accession number UDP42194.1) and E. lactis (GenBank accession number WP_156271834.1), with different nucleotide patterns. Elts, E. lactis.

FIG 2

Maximum likelihood phylogenetic tree of 274 E. lactis genomes based on core genome alignment. Different isolation origins are classified as follows: human clinical samples in red, human colonization samples (stool, gastrointestinal, genitourinary, and breast milk samples) in brown, human samples of undetermined origin in gray, animal isolates in green, food samples in yellow, dairy samples in magenta, probiotic samples in blue, environmental samples in purple, and samples with other origins (unknown) in black. No clear patterns among isolates from different sources are visible. Clinical E. lactis samples are clustered among probiotic/dairy and animal samples.