Introduction

Enterococcal genomics is a rapidly growing area of study. The first enterococcal genome sequence—that of Enterococcus faecalis V583—was published ten years ago (McShan & Shankar, 2002; Paulsen, et al., 2003), and complete or draft genome sequences of various enterococcal strains and species now number in the hundreds (http://www.ncbi.nlm.nih.gov/genome). Concurrent with rapid advances in genome sequencing, the sequencing-based classification scheme of multilocus sequence typing (MLST) has been used to interrogate population structures of the two enterococcal species that are most associated with human health and disease, E. faecalis and Enterococcus faecium. These two species also constitute the bulk of enterococcal genome sequence data that has been generated to date. This wealth of genomic data has allowed for an investigation of enterococcal diversity at a depth not previously achievable. Genomic studies in enterococci have been driven by overarching questions, such as: Why do multiple species of enterococci exist that inhabit seemingly identical niches, such as E. faecalis and E. faecium in the human gut, and what ecological factors have contributed to their divergence from a common ancestor? Within an enterococcal species such as E. faecalis or E. faecium, what qualities distinguish one strain from another? Are infection- or hospital-derived strains evolutionarily distinct from strains that benignly co-exist in the complex microbial consortium of the healthy human intestine? Related to this, have antibiotic use and the nosocomial environment led to changes in the enterococcal genome and/or its population structure?

This chapter highlights major advances in enterococcal genomics, including the development of MLST schemes to study the population structures of E. faecalis and E. faecium; comparative genome hybridization (CGH) studies to catalog the genomic contents of hundreds of E. faecalis and E. faecium strains; and significant findings from genome sequencing of multiple enterococcal species, beginning with the discovery and sequencing of the E. faecalis pathogenicity island (PAI). Additionally, we review the use of genome resequencing as a tool to study the short-term evolution of E. faecalis and the use of metagenomics to assemble in situ enterococcal genomes. In concluding the chapter, we discuss future perspectives in enterococcal genomics, including pressing questions that should drive future research in this field. While comparative genomics in enterococci has rapidly advanced over the last ten years, the number of genomes discussed here pales in comparison to what has been emerging—136 enterococcal genomes have been sequenced as part of the Human Microbiome Project (http://www.hmpdacc.org/), and 406 more were sequenced in a large-scale enterococcal genome sequencing endeavor performed in a multi-national collaboration with the Broad Institute (Cambridge, MA). Clearly, our foray into enterococcal genomics has only just begun.

Population structures of E. faecalis and E. faecium defined by MLST

MLST is a sequencing-based typing method that was designed to infer the global and long-term epidemiology of a particular bacterial species. In addition, MLST has been widely used in both population and evolutionary biology (Urwin & Maiden, 2003). The technique relies upon the amplification and sequencing of internal fragments of multiple housekeeping genes that are present at different locations on a chromosome. MLST is similar in concept to 16S rRNA sequencing, in that both techniques rely upon the sequencing of genomic loci for which diversifying selection is low, but is distinguished from 16S rRNA sequencing in its ability to resolve relationships at a subspecies level. Although MLST is able to infer bacterial population structure using hundreds or thousands of strains, one of its drawbacks is the limited number of alleles that are analyzed—only seven for E. faecium and E. faecalis—which are presumed to represent the evolutionary history of the entire genome. This limitation can be overcome by comparing full genome sequences, as has been shown by studies of the pathogens Staphylococcus aureus and Streptococcus pneumoniae, in which the recent evolution of pathogenic lineages was described by the analysis of several dozen genome sequences (Croucher, et al., 2011; Harris, et al., 2010). However, as a facile classification method, MLST has shown that certain lineages within E. faecalis and E. faecium are consistently associated with infections, multidrug resistance, and hospital persistence, and this information has helped guide the selection of strains for genome sequencing projects.

Early Comparative Genome Analysis Reveals the Existence of a Pathogenicity Island in E. faecalis

In a retrospective study of an outbreak of 206 enterococcal bacteremias at the University of Wisconsin Hospitals and Clinics over a 17-month period, 190 were found to be caused by E. faecalis, most of which were resistant to high levels of aminoglycosides and macrolides (Huycke, Spiegel, & Gilmore, 1991). Moreover, nearly half of the 190 infections were caused by a single E. faecalis lineage, as defined by pulsed-field electrophoresis (PFGE) patterns (now known to be ST6/CC2 (McBride, Fischetti, Leblanc, Moellering, Jr., & Gilmore, 2007)), which was multidrug resistant as well as hemolytic, whereas the remainder were caused by largely non-hemolytic idiosyncratic strains with few identities by PFGE (Huycke, Spiegel, & Gilmore, 1991). This indicated that one strain, termed MMH594, had become highly hospital-adapted, antibiotic resistant, and unusually pathogenic. Nearly simultaneously, the first vancomycin-resistant Enterococcus in the United States was isolated at Barnes-Jewish Hospital at Washington University (Sahm, et al., 1989). This strain, V583 (now also known to be ST6/CC2 (McBride, Fischetti, Leblanc, Moellering, Jr., & Gilmore, 2007)) was identified as E. faecalis, and represented a novel vancomycin resistance phenotype, termed VanB. Identical VanB strains were isolated over a span of three months from the bloodstream, urine, and feces of a chronically infected patient who had received vancomycin therapy prior to the first isolation (Sahm, et al., 1989). An identical isolate was later obtained from another patient in the same ICU, but who had not received vancomycin therapy, and identical isolates were obtained from urine and blood over the course of two weeks (Sahm, et al., 1989).

In examining the genomes of the multidrug-resistant hospital outbreak strain from Wisconsin and the vancomycin-resistant strains from St. Louis by sequence analysis, a number of new traits for enterococci were discovered and found to be common to the hospital strains from both locations. These included a capsule (Hancock & Gilmore, 2002; Hancock, Shepard, & Gilmore, 2003), a novel adhesin termed enterococcal surface protein (Esp) that was enriched in clinical isolates (Shankar, et al., 2001; Shankar V. , Baghdayan, Huycke, Lindahl, & Gilmore, 1999), and a bile acid hydrolase. An operon for the enterococcal cytolysin was also found, which is a factor that confers lethality to enterococcal infection in humans (Huycke, Spiegel, & Gilmore, 1991) and animals (Chow, et al., 1993; Garsin, et al., 2001; Ike, Hashimoto, & Clewell, 1984; Ike, Hashimoto, & Clewell, 1987; Jett, Jensen, Nordquist, & Gilmore, 1992; Singh, Qin, Weinstock, & Murray, 1998); is enriched in bloodstream E. faecalis isolate collections (Huycke & Gilmore, 1995; Huycke, Spiegel, & Gilmore, 1991; Ike, Hashimoto, & Clewell, 1987); is novel in structure (Bogie, Hancock, & Gilmore, 1995; Booth, et al., 1996; Coburn & Gilmore, 2003; Coburn, Hancock, Booth, & Gilmore, 1999; Cox, Coburn, & Gilmore, 2005; Gilmore, Segarra, & Booth, An HlyB-type function is required for expression of the Enterococcus faecalis hemolysin/bacteriocin, 1990); and is regulated by a novel quorum-sensing mechanism (Haas, Shepard, & Gilmore, 2002) that enables enterococci to detect target cells at a distance (Coburn, Pillar, Jett, Haas, & Gilmore, 2004). Aggregation substance, an enterococcal surface factor that causes clumping, was first described in the Clewell lab (Yagi, et al., 1983), and was shown to be lethally synergistic with the cytolysin in endocarditis (Chow, et al., 1993), was also present in the genomes of these clinical isolates. Intriguingly, all of these factors, except the capsule, occurred on a single macrorestriction fragment (Shankar, Baghdayan, & Gilmore, 2002). Further sequence analysis revealed that these traits were organized into an island of over 150 kb in size that included a phage-like integrase and excisionase, differed in G+C content from the rest of the chromosome, possessed flanking 10 bp direct repeats, and was inserted at a lysyl-tRNA locus (Shankar, Baghdayan, & Gilmore, 2002), which was reminiscent of the organization of PAIs in Gram-negative bacteria (Langille, Hsiao, & Brinkman, 2010). The E. faecalis PAI was the first canonical PAI to be identified in a Gram-positive bacterium.

Compared to those occurring in V583 and V586, the 153,571 bp PAI of strain MMH594 was found to be closest to the prototype, in that the islands from the vancomycin-resistant St. Louis strains V583 and V586 from the chronically infected patient possessed additional IS elements (IS256 and IS905) that disrupted the cytolysin operon (Shankar, Baghdayan, & Gilmore, 2002) (Figure 1). Moreover, these IS elements volatilized this region of the island, which resulted in a 17 kb deletion that distinguishes V583 from V586 and occurs at a very high frequency of 1 in 10³ V586 cells (Shankar, Baghdayan, & Gilmore, 2002). This indicates that the chronically infected St. Louis HIV/AIDS patient was infected with a mixed population of V583/V586 cells. Adaptation of the microbe in chronic infection, as seen with V583/V586, is similar to that seen with P. aeruginosa in chronically infected cystic fibrosis lungs (Hoboth, et al., 2009). Thus, from comparative sequence analysis and in vitro experiments, it is clear that the PAI is a dynamic component of E. faecalis genomes, and rapid changes in its structure are likely to impact E. faecalis virulence (Figure 1).

Figure 1.

A dynamic PAI in hospital-adapted E. faecalis. Alignment of PAI sequences from hospital infection isolates E. faecalis MMH594, V586, and V583. (A) Overall comparison of PAIs reveals expansion of the prototypical MMH594 PAI by IS acquisition (indicated (more...)

Further studies have found that the PAI has a modular structure and likely evolves by accretion of modules obtained through horizontal gene transfer (McBride, et al., 2009). A collection of 53 esp⁺ E. faecalis strains were interrogated for the presence of MMH594 PAI genes using dot blot hybridization, PCR, and a novel pathoarray with probes that corresponded to each of the 129 open reading frames of the MMH594 PAI. Presence of esp was chosen as a marker for PAI presence in the 53 strains, as esp in E. faecalis had only been detected within the PAI. Incongruence between phylogenies built from MLST alleles and from PAI gene content in the 53 strains suggested that the PAI and the core E. faecalis genome evolve through different mechanisms. Transversal clustering of PAI hybridization data revealed that the canonical PAI contains six modules (named A through F, moving from 5ʹʹ to 3ʹ PAI ends) of consecutive genes (with the exception of module E) that appear to be acquired or inherited as discrete units. Module A is composed of the pAM373-like plasmid remnant that is present at the 5ʹ end of the MMH594 PAI (PAI ORFs EF0005-EF0033; (Shankar, Baghdayan, & Gilmore, 2002)). That this module has high identity to a pheromone-responsive plasmid provides a straightforward mechanism for its horizontal transfer. It is flanked on one end by a putative phage integrase and excionase and by transposases on the other end, which could also contribute to the mobility of this module. Module B (EF0042-EF0049) contains the cytolysin operon and occurs 3ʹ to a conjugated bile salt hydrolase gene that is flanked by transposases (EF0039-EF0041). Module C (EF0051-EF0074) possesses esp and the gls24-like gene, among others, as well as an internal recombinase gene and flanking transposase (EF0075). This transposase also flanks Module D (EF0076-EF0092), which is composed of sugar uptake and metabolism genes. Module E is non-contiguous (EF0093-EF0108 and EF0124-EF0128), and encodes various genes that are likely involved in metal transport and cofactor biosynthesis. Module F (EF0108-EF0122) is internal to Module E and possesses several transposases that may provide mobility to this module.

Collectively, these data suggest that the PAI evolves by the accretion of discrete modules bounded by transposases, and is transmitted by horizontal gene transfer, as opposed to a model where a single progenitor PAI entered an ancestral E. faecalis strain and was diversified by differential gene loss in diverging MLST lineages. If an evolution-by-accretion model is correct, we could expect to discover novel PAI modules in comparative genome analyses. Consistent with this proposal, a novel PAI module has been identified in genomes of the CC2 strains HH22 and TX0104 (Solheim, et al., 2011). This module encodes several hypothetical proteins and a predicted mucin-binding domain protein (Solheim, et al., 2011), and occurs between MMH594 PAI ORFs EF0040 and EF0065, which correspond to Module B and part of Module C as defined above. Despite its absence in MMH594 and V583 genomes, the presence of this module was found to be enriched among CC2 strains, compared to non-CC2 strains, in a PCR-based screening analysis (Solheim, et al., 2011). This result suggests that comparative genomics will reveal much about the module diversity and evolutionary history of the PAI. Indeed, one E. faecalis strain for which genome sequence is available, T1, possesses a ~60 kb PAI located at the same PAI att site, which is composed entirely of novel modules not present in the prototypical MMH594 PAI (Palmer and Gilmore, unpublished).

The First Complete Enterococcal Genome, E. faecalis V583, and Comparison With OG1RF

E. faecalis V583 was provided to TIGR in 1998 and its genome sequence was published in 2003 (Paulsen, et al., 2003). The release of this genome was significant for many reasons, including its provision of a complete, high-quality reference for subsequent genome projects. V583 was selected because it was the first vancomycin-resistant Enterococcus isolated in the United States, and as noted above, was found to be similar to MMH594 and representative of the ST6/CC2 lineage (McBride, Fischetti, Leblanc, Moellering, Jr., & Gilmore, 2007; Sahm, et al., 1989). A significant and striking finding of the V583 genome is the amount of mobile DNA contained within it—calculated to be over 25% of the genomic content (Paulsen, et al., 2003). This includes seven predicted prophages, multiple integrated plasmids, IS elements and genomic islands (including the PAI), a vanB-type transposon that confers vancomycin resistance, and three extrachromosomal plasmids (pTEF1, pTEF2, and pTEF3), including one that encodes multiple antibiotic resistance genes (pTEF1) (Lepage, et al., 2006; McBride, Fischetti, Leblanc, Moellering, Jr., & Gilmore, 2007; Paulsen, et al., 2003) (Figure 2). Two of the pTEF plasmids, pTEF1 (66.3 kb) and pTEF2 (57.7 kb), are predicted pheromone-responsive plasmids similar to the extensively studied pheromone-responsive plasmids pAD1 (Clewell, 2007) and pCF10 (Dunny, 2007), respectively (see Extrachromosomal and Mobile Elements in Enteroocci). These plasmids detect small peptides produced by plasmid-free cells to induce high-efficiency transfer (transfer frequencies of up to one transconjugant per 10–100 donors; (Hirt, Schlievert, & Dunny, 2002; Huycke, Gilmore, Jett, & Booth, 1992; Licht, Laugesen, Jensen, & Jacobsen, 2002)), and their replication mechanisms appear to be functional only in the faecalis species (Palmer, Kos, & Gilmore, 2010). pTEF3 (18.0 kb) possesses the broad host range Inc18 replicon of pAMβ1 (Paulsen, et al., 2003). The remarkable amount of horizontally acquired material in the V583 genome leads to the proposition that enterococci have a special propensity to acquire and disseminate mobile elements, such as those that encode antibiotic resistance genes (Paulsen, et al., 2003).

Figure 2.

E. faecalis genome plot. The outermost ring shows E. faecalis V583 chromosomal (scaffold 4) and plasmid scaffolds (scaffold 1, pTEF2; scaffold 2, pTEF3; scaffold 3, pTEF1), with each V583 gene represented as a radial position along the ring. The locations (more...)

A second complete E. faecalis genome was published in 2008 (Bourgogne, et al., 2008), which provides a comparator to V583. E. faecalis OG1RF is a laboratory-generated rifampicin and fusidic acid-resistant derivative of the non-antibiotic resistant human oral isolate OG1 (Gold, Jordan, & van Houte, 1975), which was originally isolated prior to 1975. OG1RF is an ST1 strain, and is not a member of any of the high-risk E. faecalis MLST lineages. Compared to V583, the OG1RF genome has little mobile content, lacks plasmids and the PAI, and possesses only one prophage that is core to the faecalis species (prophage 2; (McBride, Fischetti, Leblanc, Moellering, Jr., & Gilmore, 2007)), as well as a putative transposable element that lacks antibiotic resistance genes (Bourgogne, et al., 2008). The abundance of horizontally acquired DNA in V583, as compared to OG1RF, results in a large size discrepancy between their genomes: 3.36 Mb for V583, compared to 2.74 Mb for OG1RF. Interestingly, despite the 620 kb of additional coding potential in the V583 genome, including the PAI, OG1RF can cause disease in animal models of infection (Bourgogne, et al., 2008), which highlights the point that even commensal strains of E. faecalis can be opportunists, and that basic fitness functions are important to the ability of members of this species (and probably genus) to cause disease.

Dynamic E. faecalis Genomes and CRISPR-Cas Defense

Further genome sequencing has illuminated the extent of mobile content and range of genome sizes in the faecalis species. A third complete E. faecalis genome has been announced (Brede, Snipen, Ussery, Nederbragt, & Nes, 2011), although a detailed comparative analysis has not yet been published. E. faecalis 62 was isolated from the feces of a healthy human infant and is a ST66 strain, which is a lineage that has not been associated with either infection or hospitals (Solheim, Aakra, Snipen, Brede, & Nes, 2009; Solheim, et al., 2011). The extrachromosomal elements of this strain, and to some extent the PAI, have been described (Brede, Snipen, Ussery, Nederbragt, & Nes, 2011), and their abundance and presence, respectively, indicate that the E. faecalis 62 genome is more similar to that of V583 than OG1RF in its mobile genome content. E. faecalis 62 possesses three predicted plasmids—one similar to the cryptic plasmid pS86 (EF62pA; 5.1 kb) and two putative pheromone-responsive plasmids that are similar to pCF10 (EF62pB; 51.1 kb) and pAM373 (EF62pC; 55.4 kb) (Brede, Snipen, Ussery, Nederbragt, & Nes, 2011). A pseudotemperate linear phage (EF62φ; 30.5 kb) is also present, as is a chromosomally encoded Tn916 and a variant PAI encoding esp (Brede, Snipen, Ussery, Nederbragt, & Nes, 2011). Collectively, this results in a genome size of 3.13 Mb, which is intermediate between those of V583 and OG1RF.

Sixteen additional E. faecalis genomes were sequenced and assembled to high-quality draft status as part of a enterococcal genome sequencing project that increased the amount of available sequencing data for the genus by approximately 10-fold (Palmer, et al., 2010). The 16 E. faecalis strains were selected to represent the deepest nodes in the faecalis MLST phylogeny, and were isolated over an approximately 80-year period from clinical and non-clinical sites around the world (McBride, Fischetti, Leblanc, Moellering, Jr., & Gilmore, 2007). Analysis of the 16 draft E. faecalis genomes has further supported the proposition that mobile element acquisition is a major contributor to genome diversity in the faecalis species. The genome sizes of all 16 strains are intermediate between the extremes of V583 and OG1RF, with multidrug-resistant strains generally possessing larger genomes (Palmer, et al., 2012). Strains with genomes larger than 3 Mb encode significantly more protein domains associated with mobile elements, including plasmid replication initiation and mobilization domains, a plasmid addiction toxin domain, a transposase domain, and the anti-restriction protein ArdA domain, which is encoded by the self-mobilizable transposon Tn916 (Palmer, et al., 2012). This is consistent with these strains acquiring foreign genetic elements conferring new traits, which results in larger genomes. The observed range of genome sizes suggests that the propensity to acquire mobile elements in situ varies within the faecalis species.

Genome size and the extent of acquired antibiotic resistance appear to be related to the CRISPR-cas status of E. faecalis strains. Clustered, regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (cas) genes provide a type of acquired immune defense in prokaryotes (Wiedenheft, Sternberg, & Doudna, 2012). CRISPR-Cas systems have been shown to block phage infections and plasmid uptake, and thus are endogenous barriers to horizontal gene transfer (see Enterococcal bacteriophages and genome defense). In the comparative analysis of V583 and OG1RF, it was noted that OG1RF possesses CRISPR-cas while V583 lacks it, which suggests that CRISPR-Cas defense prevented the accumulation of mobile genetic elements in OG1 prior to its isolation from the human mouth (Bourgogne, et al., 2008). Supporting a role for CRISPR-Cas systems in E. faecalis genome defense, genome size distributions significantly differ between E. faecalis strains that possess or lack CRISPR-cas, with strains that lack CRISPR-cas having larger genomes (with an average size of 3.1 Mb, compared to 2.9 Mb for strains that possess CRISPR-cas) (Palmer, et al., 2012). Similarly, strains that lack CRISPR-cas in their genomes encode significantly more conserved protein domains associated with mobile elements (Palmer, et al., unpublished). Analysis of 48 E. faecalis strains using comparative genomics and a PCR-based screening approach found a significant relationship between acquired antibiotic resistance and CRISPR-cas, with strains that lacked CRISPR-cas possessing more acquired antibiotic-resistance genes (Palmer & Gilmore, 2010). Strains deficient for CRISPR-cas are additionally enriched for cytolysin and aggregation-substance genes (Lindenstrauss, et al., 2011), both of which are encoded on pheromone-responsive plasmids and the PAI. Finally, all CC2 strains characterized thus far lack CRISPR-cas (Palmer & Gilmore, 2010), which suggests that the absence of this line of defense is a core property of the lineage that perhaps contributes to its success in acquiring novel genomic content. The role of CRISPR-Cas defense in regulating the influx of mobile elements into E. faecalis genomes is of high interest.

The Recombinogenic Nature of E. faecalis

Genomics has illuminated the extent of recombination in E. faecalis. As might be expected from MLST analyses, the inter-relationships of 18 E. faecalis strains (16 draft genomes discussed above, along with V583 and OG1RF) could not be confidently resolved by a phylogeny built from 847 core gene sequences (Palmer, et al., 2012). In pairwise comparisons of the same 18 strains, the average nucleotide identity in shared genes varied little (97.8-99.5% average identity). However, total shared gene content varied widely, which is likely a function of the varying genome sizes of the strains (Palmer, et al., 2012). This is consistent with E. faecalis strains generally being closely related in their core genes, with most variation introduced through the acquisition of horizontally transferred material. That said, signatures of recombination are apparent in some E. faecalis genomes (Fig. 2). Three E. faecalis strains, JH1 (ST40), Merz96 (ST103), and T2 (ST11), appear to have acquired genomic islands (the PAI, efaB5, and a putative genomic island that flanks the V583 vanB transposon, respectively), as well as a sequence adjacent to the islands, from strains closely related to V583 (Palmer, et al., 2012). The recombinant region of Merz96 overlaps the fsr locus, of interest because variation in the fsr region influences production of gelatinase (42). The Merz96 fsr region was previously shown to be similar to that present in V583, MMH594, and HH22—all ST6 strains (Galloway-Peña, Bourgogne, Qin, & Murray, 2011). It is likely that Merz96 acquired this region, as well as efaB5, through recombination with a ST6 strain.

A mechanism for recombinatorial transfer of large chromosomal regions between E. faecalis strains has been elucidated, and provides an explanation for decades of observations that implicate chromosomal DNA exchange in enterococci (Manson, Hancock, & Gilmore, 2010). In laboratory mating experiments with V583 as donor and OG1RF as recipient, hybrid V583-OG1RF transconjugants were observed that possessed up to 857 kb of the V583 genomic sequence. Depending on the location of the selectable marker in the V583 donor genome, transconjugants were observed that possessed the entire PAI, the V583 capsule biosynthesis operon, the V583 vancomycin resistance operon, and/or V583 gdh and yqiL MLST markers, which generated a double locus MLST variant of OG1RF in one mating experiment (Manson, Hancock, & Gilmore, 2010). Displacement of the OG1RF CRISPR-cas locus by V583 sequence was also observed (Palmer & Gilmore, 2010). Transfer of large chromosomal regions was dependent upon the presence and transfer functions of either of the pheromone-responsive plasmids pTEF1 or pTEF2 (Manson, Hancock, & Gilmore, 2010). Both pTEF1 and pTEF2 were found to integrate into the chromosome at shared IS256 sequences, and from there, likely initiated transfer induced by OG1RF pheromones, which resulted in mobilization of the V583 chromosome. The PAI and efaB5 appear to be hotspots for IS element accumulation, and IS elements occur in the genomic island that surrounds vanB (Paulsen, et al., 2003), which suggests that pheromone-responsive plasmids that possess similar IS elements could readily integrate into these regions. It is likely that Merz96, JH1, and T2 have acquired genomic islands, along with surrounding sequences, by a similar mechanism. This pheromone-responsive plasmid-dependent chromosome transfer mechanism likely contributes to the recombinogenic population structure of the faecalis species.

E. faecium Genome Sequencing

Table 1 provides an overview of several important characteristics of the first 31 publicly available E. faecium genomes. Most (23/31) of these isolates originated from human infection or from hospitalized patients that were colonized by antibiotic resistant strains. Six genomes are from strains that were isolated from feces of healthy humans (and likely represent human commensal isolates). E. faecium strains from non-human sources are poorly represented in this first set of E. faecium genomes, with genomes of only two representative strains sequenced. Both strains had been isolated from dog feces (de Regt, et al., 2012). There is a large sample bias toward clinical isolates in E. faecium strains that have been selected for genome sequencing studies. However, clinical isolates form only a minute fraction of the total E. faecium population, and it is important that genomes of E. faecium strains from non-human sources be included in sequencing projects. Another bias that is evident from Table 1 is the overrepresentation of strains from Europe and North America. In fact, only three strains (a clinical isolate from Brazil and a commensal and a clinical isolate from Australia) do not originate from these two continents. More balance is needed to fully understand the global diversity of E. faecium.

Table 1.

Initial 31 publicly available E. faecium genome sequences

The first completely finished E. faecium genome sequence became available in March 2012 and was determined for strain Aus0004. Aus0004 is a vancomycin-resistant strain that was isolated from the bloodstream of a patient in Melbourne, Australia, in 1998 (Lam, et al., 2012). The Aus0004 genome sequence was determined by a combination of 454 and Illumina sequencing and was finished by primer walking. A bacterial artificial chromosome (BAC) library was prepared to resolve chromosomal duplications. A second complete genome sequence was determined for strain TX16 (also named TX0016 or DO), which was isolated from the blood of a patient with endocarditis in 1992. Unassembled fragmentary sequences were first sequenced by Sanger sequencing in 1999, and were completed with additional 454 sequencing and PCR to close gaps (Qin, et al., 2012).

Other draft E. faecium genomes have been generated by either 454 or Illumina sequencing. Most fragmentary assemblies are a consequence of the large number of repetitive elements in the genomes of E. faecium, which are challenging to resolve bioinformatically. These elements include IS elements (for example, there are 95 IS elements in Aus0004 [77]), but also repeats within protein coding sequences, such as those that are present in the gene that encodes the Esp surface protein (Leavis, et al., 2004). Practically all E. faecium strains with available genome sequences were isolated in the last 20 years. The oldest strain is E1636, which was isolated in 1961 from a bloodstream infection in the Netherlands. Interestingly, this strain encodes the tetracycline resistance gene tetM (van Schaik, et al., 2010), though this antibiotic had only been on the market since 1952. It would be of particular interest to sequence the genomes of E. faecium strains from the pre-antibiotic era, as this would definitively answer the question if E. faecium strains are a natural source of antibiotic-resistance genes or have acquired all their resistances through lateral gene transfer in the last 70 years. While limitations exist for the first available set of E. faecium genome sequences, these data have provided us with important new insights into the evolution and basic biology of this organism.

Toward a Genome-Based Reconstruction of E. faecium Evolution

In the first genome-based study of E. faecium, seven isolates were sequenced (148). Based on a phylogenetic analysis of the concatenated alignments of 649 protein sequences, it was shown that the human commensal strain E980 was relatively distantly related to the other six E. faecium isolates, which suggested the presence of a deeply branching division within the species. Interestingly, a similar division between two major clades within E. faecium had previously been suggested by non-sequence based studies, in which random amplified polymorphic DNA (RAPD)-PCR, AFLP, and PFGE were used to characterize a collection of E. faecium strains (Vancanneyt, et al., 2002; Vankerckhoven, et al., 2008).

A divide between two major clades in E. faecium was also found by analyses of MLST data and additional genome sequence-based studies (Galloway-Peña, Roh, Latorre, Qin, & Murray, 2012; Galloway-Peña, Rice, & Murray, 2011; Palmer, et al., 2012). BAPS analysis of MLST data (Willems, et al., 2012), combined with genome sequence data, confirmed a major split in the E. faecium population (Figure 3). To further analyze the differences between the two clades, termed A and B, Palmer et al. (Palmer, et al., 2012) performed pairwise comparisons of 8 E. faecium genomes to determine the average nucleotide identity (ANI) in genes shared between the genomes. Pairwise genome comparisons between clade A and clade B strains revealed ANI values that ranged from 93.9 to 95.6%. In examining other species, ANI values of 95 ± 0.5% have been found to correlate with 70% DNA-DNA hybridization values, which is the threshold usually used to distinguish different species (50). By this criterion, clade A and clade B would represent separate species-level subdivisions of E. faecium. A recent study including 51 newly sequenced E. faecium genomes (Lebreton, et al., 2013) provided evidence for a second split in the E. faecium population, which distinguishes modern clinical isolates (clade A1) from most animal-derived strains (clade A2) in the dataset. After removing recombined regions in the genomes, mutation rates were calculated using the BEAST algorithm (Drummond, Suchard, Xie, & Rambaut, 2012) for the three clades (A1, A2 and B) of the E. faecium population. Interestingly, a significantly higher mutation rate was found for clade A1 strains (4.9 × 10⁻⁵ ± 0.3 × 10⁻⁵ substitutions per nucleotide per year) than for both clade A2 (3.6 × 10⁻⁶ ± 0.6 × 10⁻⁶ substitutions per nucleotide per year) and clade B (1.3 × 10⁻⁵ ± 0.2 × 10⁻⁵ substitutions per nucleotide per year). The inferred mutation rates for each clade were used to estimate the time of divergence between the different E. faecium clades A1, A2, and B, placing the time of the split between clade A and B at 2,776 ± 818 years ago and the split between clade A1 and clade A2 at 74 ± 30 years ago (Lebreton, et al., 2013).

Figure 3.

Phylogenomic analysis of E. faecium. Minimum evolution tree based on the concatenated alignments of 274 orthologous proteins conserved in draft genome sequences of 30 E. faecium isolates. Bootstrap values are indicated and are based on 1000 permutations. (more...)

Human commensal isolates overwhelmingly belong to clade B (which corresponds to BAPS-group 1 defined by Willems et al. (Willems, et al., 2012)), while strains from other sources, including hospital-acquired infections and animal isolates, cluster in clade A1 and A2, respectively (Lebreton, et al., 2013). However, this ecological distinction between two clades is not absolute: of the strains with sequenced genomes, strain 1,141,733 was isolated from a bloodstream infection but clusters in clade B, while strain E1039, which was isolated from the feces of a healthy volunteer, clusters in clade A. Notably, the presence of evolutionarily distinct clades within what we currently characterize as E. faecium is very different from what genomics has revealed for E. faecalis, where isolates can be so closely related in core genes as to be virtually indistinguishable from one another.

Recently, a lack of CRISPR-Cas defense has been implicated in the evolution of multidrug-resistant E. faecalis (Palmer & Gilmore, 2010). CRISPR-Cas systems appear to be relatively rare in E. faecium. Among the first 31 publicly available E. faecium genomes, a CRISPR-Cas system can be identified in only five isolates (strains TX1330, Com12, 1,141,733, PC4.1, and 1,231,408) (Palmer & Gilmore, 2010) and unpublished observations). Except for strain 1,231,408, each of these strains belongs to clade B; strain 1,231,408 is a hybrid clade A-clade B strain that appears to have acquired CRISPR-cas through recombination with a clade B strain (Palmer, et al., 2012). It has been hypothesized that the absence of CRISPR-Cas in E. faecium would allow for the facile acquisition of foreign DNA, such as plasmids that encode genes for antibiotic resistance (Palmer & Gilmore, 2010). However, even commensal E. faecium isolates with very few resistances (such as E1039 and E980) lack a CRISPR-Cas system, which indicates that the absence of CRISPR-Cas does not necessarily lead to the rapid accumulation of antibiotic resistance genes (van Schaik, et al., 2010). Further genome sequencing of antibiotic-resistant and antibiotic-sensitive isolates is required to determine if CRISPR-Cas defense contributes significantly to the evolution of multidrug resistance in E. faecium. Nevertheless, based on available genome data, it appears that CRISPR-Cas defense is an ancestral aspect of the clade B E. faecium lineage and not of clade A.

Intraspecies Diversity of E. faecium

Bacterial genomes differ as the result of drift in the sequences of shared genes (with some mutations becoming fixed by selection), as well as from the acquisition of new genes by horizontal gene transfer. A first insight into variation in gene content of different E. faecium isolates was provided by a study in which comparative genomic hybridization was used to characterize the accessory gene pool of E. faecium (Leavis H. L., et al., 2007). This led to the identification of 175 genes found more frequently in clinical isolates than in non-clinical isolates. This approach identified the transposon IS16 as most specifically enriched in clinical isolates (Leavis H. L., et al., 2007). This study also showed that, based on gene content, hospital isolates were more similar to each other than to isolates from other sources, such as healthy humans and animals.

Clinical E. faecium isolates are predominantly found within clade A1, and these appear to have emerged from a background of animal isolates, while human commensal isolates are more distantly related (Lebreton, et al., 2013). Genome sequence analysis of two ampicillin-resistant strains (E4452 and E4453) that were isolated from dogs (de Regt, et al., 2012) appear to corroborate the hypothesis that strains from lineage-78 in clade A1 have evolved from animal isolates, since strains E4452 (ST266) and E4453 (ST192) both belong to lineage-78, as defined in the previously described BAPS analysis of E. faecium MLST data (159). Phylogenomic analyses underscore the relatively close relationship of E4453 with strains that belong to either lineage-78 or lineage-18 (de Regt, et al., 2012). Both these canine E. faecium strains carry IS16, which was previously shown to be highly enriched among clinical isolates; but lack virulence genes, such as hyl or esp, which are commonly found in clinical isolates. Strain E4453, but not strain E4452, carries a genomic island that is putatively involved in the breakdown of complex sugars (Heikens, van Schaik, Leavis, Bonten, & Willems, 2008). However, the two canine E. faecium strains share more genes with each other than with the clinical strains E1162 and U0317, which indicates that niche adaptation is occurring on the level of the accessory genome of these isolates. For instance, both strains carry an element that is putatively involved in the breakdown and metabolism of xylopolysaccharides, which is absent from all other isolates for which the genome sequences are publicly available, and appears to be relatively rare in clinical isolates (de Regt, et al., 2012). A PCR-based analysis of the presence of this element in ampicillin-resistant strains isolated from the community showed that this element is relatively common among ampicillin-resistant E. faecium strains originating from dogs (18 positives from 25 tested strains) and cats (8 positives from 11 tested strains). This element was also found in two of only three ampicillin-resistant strains that were isolated from a total of 40 healthy volunteers who were given a week-long course of amoxicillin. All ampicillin-resistant isolates from the community were typed with MLST and found to be related to ampicillin-resistant canine E. faecium strains. The low number of ampicillin-resistant enterococci that were isolated from healthy humans underscores the relatively low colonization rate of this group by ampicillin-resistant enterococci, and indicates that ampicillin-resistant E. faecium are more frequently present in pets than in healthy humans. The finding that ampicillin-resistant isolates from pets are phylogenetically related to ampicillin-resistant clinical isolates of lineage-78 may indicate that ampicillin-resistant isolates have first emerged in pets or other animals and have acquired additional elements (such as virulence determinants, like the esp gene and transposons that confer vancomycin resistance) during their adaptation to the lifestyle of nosocomial pathogens. Further genome sequencing of ampicillin-resistant and ampicillin-sensitive strains from non-clinical and non-human reservoirs is required to clarify the evolutionary pathways that have led to the emergence of multidrug-resistant E. faecium strains that can successfully colonize and infect hospitalized patients.

To further quantify intraspecies genomic diversity and to estimate the size of the total gene pool that is accessible to a single species, pan-genome analyses are required (reviewed in (Tettelin, Riley, Cattuto, & Medini, 2008)). In these analyses, pairwise comparisons are made between all possible combinations of genome sequences of a single species, leading to estimates of the total gene pool that is accessible to the organism, the size of the core genome (namely, the number of genes that are conserved in all strains of the same species), and the number of new genes that might be expected to be discovered when additional genomes are sequenced. By this approach and by using 73 E. faecium genomes, the size of the E. faecium core genome is estimated to consist of 1597 coding sequences (Lebreton, et al., unpublished data). The total number of coding sequences in an E. faecium genome can range from 2272 (for strain EnGen0014) to 3318 (for strain TX0133a01), which indicates that between 29% and 52% of the DNA in any E. faecium isolate is non-core and is thus variably present between strains. For strain Aus0004, 38% of the genome is accessory (Lam, et al., 2012). These analyses have also found that the E. faecium pan-genome is comparatively open, which signifies that E. faecium can efficiently acquire and incorporate novel DNA into the collective gene pool of the species. This indicates that E. faecium can rapidly acquire genes that contribute to fitness, which enables it to inhabit a wide variety of environmental niches.

Diversity in the E. faecium Accessory Genome: Plasmids, Phage and Genomic Islands

Genome sequencing has shown that plasmids, prophages, and genomic islands are important drivers of diversity in the accessory genome of E. faecium.

Genomic islands

Similar to what was found in E. faecalis (Schwarz, Perreten, & Teuber, 2001), genome sequencing of E. faecium led to the identification of two genomic islands that are enriched in clinical E. faecium isolates. The most thoroughly studied genomic island in E. faecium carries the esp gene, which encodes a large (~200 kDa) peptidoglycan-anchored surface protein that contributes to biofilm formation (Heikens, Bonten, & Willems, 2007). In animal models, it has been shown to contribute to urinary tract infection (Leendertse, et al., 2009) and endocarditis (Heikens, et al., 2011), but not to gastrointestinal colonization in the model tested (Heikens, et al., 2009). In E. faecium, the esp gene is part of a large genomic island that ranges from 64 to 125 kbp in size (Figure 4), and which has been sequenced to completion in the strains E1162, E1679, U0317, and Aus0004 (Lam, et al., 2012; van Schaik, et al., 2010). As in E. faecalis (124), the esp genomic island in E. faecium carries all the hallmarks of a prototypical genomic island, as its %G+C content is 1.1% to 2.3% lower than the entire genome of this strain, and it is flanked by two 54-bp direct repeats. In all strains, the esp genomic island has been integrated between a small gene that encodes a hypothetical protein and the rpsI gene, which encodes a small ribosomal protein. In strain U0317, a genomic rearrangement event appears to have occurred after the initial integration of the esp genomic island, which resulted in the nearly complete island reintegrating 5ʹ of the tuf gene (van Schaik, et al., 2010). The locus in which the esp island integrates appears to be a hotspot for the integration of foreign elements; in several strains that lack the esp island, smaller elements of possible phage or plasmid origin have integrated at this position.

Figure 4.

ICEEfm1 in E. faecium strains Aus0004, E1162, E1679 and U0317. The ICEEfm1 elements of strains Aus0004, E1162, E1679, and U0317 were aligned using the Artemis Comparison Tool (15). Arrows indicate coding sequences (CDS) and the direction of transcription. (more...)

In E. faecium strains E1162 and UW3308, the esp island can be mobilized and horizontally transferred to esp^- acceptor strains by conjugation (Oancea, Klare, Witte, & Werner, 2004; van Schaik, et al., 2010). The observation that the esp island is mobilizable and self-transmissible has led to the renaming of this island to the integrative conjugative element (ICE) ICEEfm1 (Top, et al., 2011). Sequencing of ICEEfm1 in different E. faecium strains has revealed that this element encodes many functions that are potentially beneficial for the bacterial cell during infection of the human host. Therefore, the conjugative spread of ICEEfm1 may significantly contribute to the recent emergence of E. faecium as a nosocomial pathogen of major importance.

The overall structure of ICEEfm1 is similar in strains E1162, E1679, U0317 and Aus0004, but several insertions and rearrangements have occurred. The 5ʹ end of ICEEfm1 consists of an element that resembles the conjugation module of the widely spread conjugative transposon Tn916. At the extreme 5ʹ end of ICEEfm1 in strains E1162, E1679, and U0317, a predicted peptidoglycan-anchored surface protein is encoded. This protein is 91% identical on the amino-acid level to the collagen adhesin EcbA (Hendrickx, et al., 2009). This gene is also present in strain Aus0004, but an additional 23.8 kb fragment is present between the gene that encodes the ecbA homolog and the 54 bp sequence that forms the outer boundary of ICEEfm1. The central part of ICEEfm1 consists of the esp gene and several other genes that have predicted roles in regulation of transcription, transport, and other functions. There is considerable heterogeneity in the esp gene among different strains, particularly in the 3ʹ end of the gene where repeats are encoded (Leavis, et al., 2004). In the 3ʹ end of ICEEfm1, a 10-kb region can be identified that is essentially identical (98% nucleotide sequence identity) to a region of the E. faecalis PAI, which also encodes esp (Shankar, Baghdayan, & Gilmore, 2002). Several of the genes in this region have a predicted role in the uptake of manganese, which is an important element for virulence in both Gram-negative and Gram-positive bacteria (Papp-Wallace & Maguire, 2006). On the extreme 3ʹ end of ICEEfm1, an integrase gene is present, which is indispensable for excision of ICEEfm1 (Top, et al., 2011). Multiple transposases are scattered throughout ICEEfm1, and the presence of other elements (such as a group II intron in strain E1162) can vary from strain to strain. The most striking difference among the currently sequenced ICEEfm1 elements is the presence of a Tn1549-like transposon (Garnier, Taourit, Glaser, Courvalin, & Galimand, 2000) in the ICEEfm1 of Aus0004. This transposon carries the vanB gene cluster, which confers resistance to vancomycin. Another similar integration in ICEEfm1 has occurred in strain E1679, where a gene cluster putatively encoding a pathway for the metabolism of inositol has been inserted. This element appears to be functional, as strain E1679 can grow on inositol as carbon source, while E. faecium strains that lack these genes cannot (van Schaik et al., unpublished data). Genome sequencing of strain 1,231,502 also revealed the presence of this inositol utilization gene cluster as part of ICEEfm1 in this strain.

A second genomic island was first identified in the draft TX16 genome, due to its aberrant dinucleotide frequency and %G+C content. The island is an 8.4 kb element that is more commonly found in clinical isolates than in strains isolated from non-clinical settings (Heikens, van Schaik, Leavis, Bonten, & Willems, 2008). This genomic island is flanked by inverted and direct repeats, contains an integrase, has an anomalous %G+C; and in addition to DO, is present in the genomes of the clinical isolates 1,230,933, 1,231,502, 1,231,410, Aus0004, C68, E1162, E1679, TX0133, TX0082, and U0317, all of which are clade A strains. The only non-clinical isolate in which this element is also found in the genome sequence is the canine E. faecium strain E4453. The functional role of this element remains to be experimentally determined, but sequence analysis points towards its role in the uptake and metabolism of complex carbohydrate substrates.

Genomics of Other Enterococci

Although most sequencing efforts have has focused on genomes of faecalis and faecium species, data are increasingly available for other enterococcal species as well. These genomes, in addition to being interesting in their own right, are important for understanding the diversity of the genus Enterococcus, and to understand the evolution of the species within this genus. Annotated and unannotated draft genome sequences are available for strains of E. casseliflavus, E. gallinarum, E. italicus, E. saccharolyticus, and E. mundtii (Magni, et al., 2012; Palmer, et al., 2010; Palmer, et al., 2012) and unpublished data in GenBank).

E. casseliflavus and E. gallinarum, historically thought to be primarily associated with plants (Mundt & Graham, 1968) and birds (Collins, Jones, Farrow, Kilpper-Balz, & Schleifer, 1984), respectively, have also been associated with life-threatening infections, such as bacteremia and meningitis (Choi, et al., 2004; de Perio, Yarnold, Warren, & Noskin, 2006; Khan & Elshafi, 2011; Prakash, Rao, & Parija, 2005). A hospital outbreak of E. gallinarum has been reported (Contreras, et al., 2008). The endogenous low-level vancomycin resistance of E. casseliflavus and E. gallinarum, combined with the intrinsic resistance of all enterococci to antibiotics such as cephalosporins (Tannock & Cook, 2002), may contribute to a rise in opportunistic infections caused by these species. Three of the sequenced E. casseliflavus genomes (EC10, EC20, and EC30) and the E. gallinarum EG2 genome derive from clinical isolates obtained from patients in the United States (Palmer, et al., 2012). E. casseliflavus ATCC 12755 (Genbank accession AEWT00000000) is a reference genome for the Human Microbiome Project. Of the remaining sequenced species, to our knowledge, only E. mundtii has been associated with human infection, and reports of these types of infection are few (de Perio, Yarnold, Warren, & Noskin, 2006; Prakash, Rao, & Parija, 2005). E. mundtii ERL1656, for which a genome sequence is available, was isolated from cowʹs milk and is a bacteriocin-producing strain that is thought to have probiotic properties (Magni, et al., 2012). E. saccharolyticus 30_1 (Genbank accession ADLY00000000) was isolated from inflamed ileum tissue taken from a patient with Crohn's disease (http://www.broadinstitute.org/annotation/genome/Enterococcus_group), and is a reference genome for the Human Microbiome Project. Finally, E. italicus DSM 15952 (Genbank accession AEPV00000000), which is also a reference genome for the Human Microbiome Project, was isolated from an Italian cheese and is the type strain for the italicus species (Fortina, Ricci, Mora, & Manachini, 2004).

Two phenotypes typically absent in E. faecalis and E. faecium have been of particular interest for clinical discrimination of enterococcal species. E. casseliflavus and E. gallinarum are distinguished from E. faecalis and E. faecium by their motility, while E. casseliflavus and E. mundtii are further distinguished by their production of a yellow pigment (Facklam, Carvalho, & Teixeira, 2002). However, variability in these phenotypes occurs, which complicates species designations when phenotypic characteristics are used (Devriese, Pot, & Collins, 1993; Tannock & Cook, 2002; Vincent, Knight, Green, Sahm, & Shlaes, 1991). The genetic basis for these traits, as well as whether they are truly species-specific, are of interest. Genomic analysis has revealed genetic bases for motility and pigment production in E. casseliflavus and E. gallinarum (Palmer, et al., 2012), among other traits.

Differences Between Enterococcal Species Revealed by Genomics

Beyond the phylogenetic classification, what does it mean to be an E. faecalis, an E. faecium, an E. casseliflavus, or another enterococcal species? Are there certain traits that define these species relative to one another? A great deal of work in the clinical microbiology of enterococci has focused on biochemical or phenotypic tests that are capable of discriminating one species from another (Facklam, Carvalho, & Teixeira, 2002). However, phenotypic variability within a species and atypical isolates complicate those analyses. Comparative genomics, through the interrogation of tens or hundreds of genomes, offers a new way to search for traits that are characteristic of a species.

Additional Genomic Applications: Metagenomics and Genome Resequencing

To this point, we have discussed comparative genomic analyses of closed and draft enterococcal genomes, each obtained by de novo sequencing of DNA obtained from an isolate grown in pure culture. Here, we discuss two additional genomic techniques that have been applied to E. faecalis: metagenomics and genome resequencing.

Conclusion and Perspectives

Genome sequencing and comparative genomics has significantly advanced our understanding of enterococcal virulence, biology, and evolution. Insights discussed here include the identification of adaptive elements that likely contribute to the diverse ecology of the enterococci, analyses illuminating the fluidity of the E. faecalis and E. faecium genomes, and the discovery that the species E. faecium is actually composed of distinct phylogenetic clades between which recombination occurs. Despite rapid and significant advances in this area, much remains to be learned from existing genome data.

For example, for currently available E. faecalis genomes, most comparative analysis has focused on recombination and horizontal gene transfer, which is precipitated by multiple lines of evidence that indicate that horizontal gene transfer is rampant in the species. However, a detailed analysis of the mobile elements that populate these genomes remains to be performed. What novel traits do these plasmids, phage, and genomic islands encode? What impact does the insertion of mobile elements have on the genome; namely, are core functions disrupted? What are the core functions of the faecalis genome, and what roles might these genes play in infection, niche colonization, and persistence? Beyond faecalis, what functions are core to the other enterococcal species for which multiple genome sequences are available—faecium and casseliflavus—and what do they tell us about what it means to be classified as one of these species? Further, what can comparative genomics tell us about phylogenetic relationships and gene exchange among genera closely related to Enterococcus, such as Melissococcus and Tetragenococcus, each of which already have sequenced representatives available in GenBank? These are a handful of questions that are meant to illustrate that we have only scratched the surface of this field.

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