Emergence of epidemic multidrug-resistant Enterococcus faecium from animal and commensal strains

Abstract

Enterococcus faecium, natively a gut commensal organism, emerged as a leading cause of multidrug-resistant hospital-acquired infection in the 1980s. As the living record of its adaptation to changes in habitat, we sequenced the genomes of 51 strains, isolated from various ecological environments, to understand how E. faecium emerged as a leading hospital pathogen. Because of the scale and diversity of the sampled strains, we were able to resolve the lineage responsible for epidemic, multidrug-resistant human infection from other strains and to measure the evolutionary distances between groups. We found that the epidemic hospital-adapted lineage is rapidly evolving and emerged approximately 75 years ago, concomitant with the introduction of antibiotics, from a population that included the majority of animal strains, and not from human commensal lines. We further found that the lineage that included most strains of animal origin diverged from the main human commensal line approximately 3,000 years ago, a time that corresponds to increasing urbanization of humans, development of hygienic practices, and domestication of animals, which we speculate contributed to their ecological separation. Each bifurcation was accompanied by the acquisition of new metabolic capabilities and colonization traits on mobile elements and the loss of function and genome remodeling associated with mobile element insertion and movement. As a result, diversity within the species, in terms of sequence divergence as well as gene content, spans a range usually associated with speciation.

Importance: Enterococci, in particular vancomycin-resistant Enterococcus faecium, recently emerged as a leading cause of hospital-acquired infection worldwide. In this study, we examined genome sequence data to understand the bacterial adaptations that accompanied this transformation from microbes that existed for eons as members of host microbiota. We observed changes in the genomes that paralleled changes in human behavior. An initial bifurcation within the species appears to have occurred at a time that corresponds to the urbanization of humans and domestication of animals, and a more recent bifurcation parallels the introduction of antibiotics in medicine and agriculture. In response to the opportunity to fill niches associated with changes in human activity, a rapidly evolving lineage emerged, a lineage responsible for the vast majority of multidrug-resistant E. faecium infections.

FIG 1

goeBURST analysis of 2,273 E. faecium entries in the E. faecium MLST database (http://efaecium.mlst.net), which can be grouped into 773 sequence types (STs) (brown circles), based upon MLST relatedness. STs included in this study are highlighted in purple.

FIG 2

RAxML SNP-based tree based on the concatenated alignments of DNA sequences of 1,344 single-copy core genes in 73 E. faecium genomes. Bootstrapping was performed with 1,000 replicates. The origins of the strains are indicated. The dates for the split between the clades, estimated by a BEAST analysis, are indicated (ya, years ago). The infectivity score reflects the number of strains of a particular ST, in the MLST database, isolated from infection. The clades are color coded as follows: clade B in dark blue, clade A1 in red, and clade A2 in gray.

FIG 3

Frequency of fosfomycin resistance was determined in triplicate for 10 randomly selected strains from each E. faecium clade (clade A1 [red], A2 [gray], and B [dark blue]). Each symbol represents the average value for one strain, and the clade average ± standard deviation (error bars) for the 10 strains per clade are indicated.

FIG 4

(A) Genome size comparison for E. faecium clade A1 (red), A2 (gray), and B (dark blue). (B and C) Pan-genome (B) and core genome (C) are shown for increasing values of the number of sequenced E. faecium genomes within each clade. Circles represent the number of new or core genes present when a particular genome is added to each subset. Black bars represent median values. The curve for the estimation of the size of the E. faecium pan-genome for each clade is a least-squares power law fit through medians. The size of the core genome within each clade was estimated by fitting an exponential curve through medians. (D) Heat map showing the enrichment in genetic mobile elements in E. faecium genomes within each clade (clade A1 [red], A2, [gray], and B [light blue]). Horizontal boxes represent strains, which are ordered within clades as in Fig. 2 (rotated 90°). The aggregate length (kb) of islands was used to compare content in each clade (ranging from 4 kb to 99 kb; median, 17 kb), whereas the numbers of putative plasmids (ranging from 0 to 9; median, 3) or phage elements (ranging from 0 to 4; median, 1) are represented. The heat map reflects the 10th percentile (light gray), 50th percentile (medium gray), and 90th percentile (black). The “×” symbol in a box indicates genome sequence for which the length of genomic islands could not be determined using the SIGI-HMM algorithm (27).

FIG 5

Summary of clade-specific antibiotic resistance genes, insertion sequences (IS), and select defenses against horizontal gene transfer. Each box represents a strain, arranged by clade as shown in Fig. 2. The “×” symbol in a box indicates genome sequence with an assembly quality that precluded identification of the indicated feature. An asterisk in a box indicates hybrid genomes that contain CRISPR-cas on recombined fragments. CRISPR and type IV restriction-modification (RM) systems are included in the miscellaneous (Misc.) category.