Enterococci are unusually well adapted for survival and persistence in a variety of adverse environments, including on inanimate surfaces in the hospital environment and at sites of infection. This intrinsic ruggedness undoubtedly played a role in providing opportunities for enterococci to interact with other overtly drug-resistant microbes and acquire additional resistances on mobile elements. The rapid rise of antimicrobial resistance among hospital-adapted enterococci has rendered hospital-acquired infections a leading therapeutic challenge. With about a quarter of a genome of additional DNA conveyed by mobile elements, there are undoubtedly many more properties that have been acquired that help enterococci persist and spread in the hospital setting and cause diseases that have yet to be defined. Much remains to be learned about these ancient and rugged microbes, particularly in the area of pathogenic mechanisms involved with human diseases.

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.

Clustered, regularly interspaced short palindromic repeats (CRISPR) provide bacteria and archaea with sequence-specific, acquired defense against plasmids and phage. Because mobile elements constitute up to 25% of the genome of multidrug-resistant (MDR) enterococci, it was of interest to examine the codistribution of CRISPR and acquired antibiotic resistance in enterococcal lineages. A database was built from 16 Enterococcus faecalis draft genome sequences to identify commonalities and polymorphisms in the location and content of CRISPR loci. With this data set, we were able to detect identities between CRISPR spacers and sequences from mobile elements, including pheromone-responsive plasmids and phage, suggesting that CRISPR regulates the flux of these elements through the E. faecalis species. Based on conserved locations of CRISPR and CRISPR- cas loci and the discovery of a new CRISPR locus with associated functional genes, CRISPR3- cas , we screened additional E. faecalis strains for CRISPR content, including isolates predating the use of antibiotics. We found a highly significant inverse correlation between the presence of a CRISPR- cas locus and acquired antibiotic resistance in E. faecalis , and examination of an additional eight E. faecium genomes yielded similar results for that species. A mechanism for CRISPR- cas loss in E. faecalis was identified. The inverse relationship between CRISPR- cas and antibiotic resistance suggests that antibiotic use inadvertently selects for enterococcal strains with compromised genome defense. IMPORTANCE For many bacteria, including the opportunistically pathogenic enterococci, antibiotic resistance is mediated by acquisition of new DNA and is frequently encoded on mobile DNA elements such as plasmids and transposons. Certain enterococcal lineages have recently emerged that are characterized by abundant mobile DNA, including numerous viruses (phage), and plasmids and transposons encoding multiple antibiotic resistances. These lineages cause hospital infection outbreaks around the world. The striking influx of mobile DNA into these lineages is in contrast to what would be expected if a self (genome)-defense system was present. Clustered, regularly interspaced short palindromic repeat (CRISPR) defense is a recently discovered mechanism of prokaryotic self-defense that provides a type of acquired immunity. Here, we find that antibiotic resistance and possession of complete CRISPR loci are inversely related and that members of recently emerged high-risk enterococcal lineages lack complete CRISPR loci. Our results suggest that antibiotic therapy inadvertently selects for enterococci with compromised genome defense.

The enterococci are Gram-positive lactic acid bacteria that inhabit the gastrointestinal tracts of diverse hosts. However, Enterococcus faecium and E. faecalis have emerged as leading causes of multidrug-resistant hospital-acquired infections. The mechanism by which a well-adapted commensal evolved into a hospital pathogen is poorly understood. In this study, we examined high-quality draft genome data for evidence of key events in the evolution of the leading causes of enterococcal infections, including E. faecalis , E. faecium , E . casseliflavus , and E . gallinarum . We characterized two clades within what is currently classified as E. faecium and identified traits characteristic of each, including variation in operons for cell wall carbohydrate and putative capsule biosynthesis. We examined the extent of recombination between the two E. faecium clades and identified two strains with mosaic genomes. We determined the underlying genetics for the defining characteristics of the motile enterococci E . casseliflavus and E . gallinarum . Further, we identified species-specific traits that could be used to advance the detection of medically relevant enterococci and their identification to the species level. IMPORTANCE The enterococci, in particular, vancomycin-resistant enterococci, have emerged as leading causes of multidrug-resistant hospital-acquired infections. In this study, we examined genome sequence data to define traits with the potential to influence host-microbe interactions and to identify sequences and biochemical functions that could form the basis for the rapid identification of enterococcal species or lineages of importance in clinical and environmental samples.