Genomic and SNP analyses demonstrate a distant separation of the hospital and community-associated clades of Enterococcus faecium

Abstract

Recent studies have pointed to the existence of two subpopulations of Enterococcus faecium, one containing primarily commensal/community-associated (CA) strains and one that contains most clinical or hospital-associated (HA) strains, including those classified by multi-locus sequence typing (MLST) as belonging to the CC17 group. The HA subpopulation more frequently has IS16, pathogenicity island(s), and plasmids or genes associated with antibiotic resistance, colonization, and/or virulence. Supporting the two clades concept, we previously found a 3-10% difference between four genes from HA-clade strains vs. CA-clade strains, including 5% difference between pbp5-R of ampicillin-resistant, HA strains and pbp5-S of ampicillin-sensitive, CA strains. To further investigate the core genome of these subpopulations, we studied 100 genes from 21 E. faecium genome sequences; our analyses of concatenated sequences, SNPs, and individual genes all identified two distinct groups. With the concatenated sequence, HA-clade strains differed by 0-1% from one another while CA clade strains differed from each other by 0-1.1%, with 3.5-4.2% difference between the two clades. While many strains had a few genes that grouped in one clade with most of their genes in the other clade, one strain had 28% of its genes in the CA clade and 72% in the HA clade, consistent with the predicted role of recombination in the evolution of E. faecium. Using estimates for Escherichia coli, molecular clock calculations using sSNP analysis indicate that these two clades may have diverged ≥1 million years ago or, using the higher mutation rate for Bacillus anthracis, ∼300,000 years ago. These data confirm the existence of two clades of E. faecium and show that the differences between the HA and CA clades occur at the core genomic level and long preceded the modern antibiotic era.

Figure 1. Position of the 100 genes analyzed on the TX16 (DO) chromosome.

The chromosomal map of the Enterococcus faecium genome of the endocarditis isolate TX16, commonly referred to as DO. The arrows represent the 100 genes chosen for clade and SNP analysis and their position on the chromosome. The red arrows depict genes transcribed in the forward orientations, and blue arrows depict genes transcribed in the reverse orientation. The general location of a few subset of the 100 genes are labeled.

Figure 2. UPGMA phylogenetic tree of the concatenated 100 gene sequence (106,818 bp).

The results are based on a pair-wise analysis of the concatenated 100 gene nucleotide sequence for each of the 21 E. faecium strains using the Poisson correction method in MEGA4.0.2 software. An UPGMA tree was constructed depicting the evolutionary distance between these sequences. The tree is drawn to scale with the branch lengths representing the evolutionary distances, the scale can be seen at the bottom of the tree.