BACKGROUND
enterococci are normal constituents of the gastro-intestinal flora of humans and other animals  <cit> .
although they only occasionally cause infections in healthy individuals, they are the third most commonly isolated gram positive organisms from hospital-associated  infections in the united states and are increasingly reported in other countries  <cit> .
enterococcal infections are often difficult to treat due to the number of antibiotics to which these organisms are resistant.
some antibiotic resistances are intrinsic, such as resistances to cephalosporins, while other antibiotic resistances are acquired through mutations or horizontal gene transfer, most notably the van systems that encode vancomycin resistance  <cit> .
several recent studies also confirmed that enterococci can transfer their resistance to even more virulent organisms, such as staphylococcus aureus <cit> .
enterococcus faecalis is the most common enterococcal species recovered from infections.
however, in the last decade, infections with enterococcus faecium have been on the rise in the united states, europe, and south america  <cit> .
in the us, isolates of e. faecium now account for ca.
35% of nosocomial enterococcal isolates identified to the species level  <cit> .
it is still not clear what has caused the ecological replacement of e. faecalis with e. faecium in the nosocomial setting, but it is speculated that the intense use of antibiotics in hospitals and the multiple antibiotic resistances of e. faecium have been major contributing factors  <cit> .
a few genes have been suggested as being virulence determinants in e. faecium due to their enrichment in clinical isolates, such as the fms or hyl genes  <cit> .
however, only three genes have been experimentally implicated to have an impact on virulence in animal models, namely esp, which has a role in biofilm, urinary tract infection, and endocarditis  <cit> ; acm, encoding a collagen binding adhesin contributing to endocarditis  <cit> ; and the ebpfm operon which encodes pili that are important in biofilm and urinary tract infection  <cit> .
in addition, conjugative transfer of a plasmid with a hyl-like gene not only conferred increased resistance to vancomycin but also increased virulence in transconjugants in the mouse peritonitis model  <cit> , and a different hyl-plasmid conferred colonization in the murine gut  <cit> .
while the gene responsible for this increase in virulence and colonization have yet to be determined, the deletion of the hyl gene did not cause attenuation in the peritonitis model  <cit> .
molecular epidemiological studies of outbreaks of e. faecium using mlst initially indicated that there was a specific lineage or genogroup of strains, designated clonal complex  <dig>  that was predominant in the hospital environment  <cit> .
other studies using pyrosequencing and whole-genome microarray subsequently indicated that, while there appeared to be a globally dispersed clade containing the vast majority of epidemic and clinical isolates which harbor a large content of accessory genes specific to this clade  <cit> , isolates associated with healthcare settings were not strictly clonally related to each other.
in particular, while cc <dig> genogroup isolates are part of the ha subpopulation, not all ha isolates are considered part of the st <dig> lineage  <cit> .
recent studies in our laboratory and others have shown large differences  in the sequence of the core genome, as well as differences in the 16-s rrna, between two different clades which were named the hospital-associated clade  and community-associated  clade strains,  <cit> .
the ha clade contains most clinical and ha-associated strains but also included strains from non-hospital origin  <cit> .
molecular studies and comprehensive comparative genomic studies of e. faecium have long been hindered by the lack of a complete genome sequence.
the tx <dig>  genome was initially sequenced at the department of energy’s joint genome institute  in walnut creek, ca.
in  <dig> in an effort to demonstrate capabilities of the sequencing technology at that time by sequencing the genome in only 1 day.
however, the genome was far from closed and the past decade has been spent on annotation, final assembly, and analyses of this genome.
recently, while this manuscript was in review, a closed e. faecium genome was published by lam et al.
using the st <dig> isolate aus <dig>  which was isolated from the bloodstream of a patient in melbourne, australia  <cit> .
in this study, we report the closed genome of the us e. faecium endocarditis isolate tx <dig> , and a comparative analysis of this strain’s genome with  <dig> other available e. faecium draft genomes  <cit> , as well as the recently published aus <dig>  <cit> .
due to the fact the tx <dig> genome has been used in multiple pathogenesis studies and is a part of the clonal group representing the majority of clinical strains globally  <cit> , the complete genome sequence of e. faecium tx <dig> will facilitate future research by providing a critical starting point for genome-wide functional studies to determine the molecular basis of pathogenesis and to further understand the evolution and molecular epidemiology of e. faecium infective strains.
RESULTS
e. faecium tx <dig> general genome features
the e. faecium tx <dig> genome consists of one chromosome and three plasmids.
the chromosome  contains  <dig> ,137 bp with  <dig>  protein-coding orfs,  <dig> trnas,  <dig> copies of ribosomal rrna and  <dig> other non-coding rnas .
the chromosome has a gc content of  <dig> %, and it shows a clear gc skew at the origin of replication .
the sizes of the three plasmids  are  <dig> ,  <dig>  and  <dig>  bp, encoding  <dig>   <dig>  and  <dig> orfs, respectively .
to investigate the conservation of the gene order of e. faecium compared to its close relative e. faecalis, a blastp alignment of all the predicted proteins from the tx <dig> and v <dig> genomes was performed followed by orf synteny analysis using dagchainer  <cit> .
the result showed that e. faecium tx <dig> gene order is very different from that of e. faecalis strain v <dig>  and all orf synteny blocks were relatively short .
interestingly, when comparing tx <dig> to the closed genome aus <dig>  which was published while this paper was in review, mauve genome alignment analysis resulted in  <dig> locally collinear blocks for both tx <dig> and aus <dig> ranging from  <dig> – <dig>  bp for tx <dig> and  <dig> – <dig>  bp for aus <dig> .
the two isolates had very similar synteny, although two regions found in tx <dig> were inverted in aus <dig>  two site-specific tyrosine family recombinases  were found flanking these two inversions .
the genome size of the e. faecium strains vary substantially from  <dig>  mb  to  <dig>  mb , while the number of orfs varies from  <dig>   to  <dig>  .
ortholog analysis of tx <dig> compared to tx <dig> and all the available but unfinished e. faecium genomes using blastp of predicted protein sequences and orthomcl resulted in  <dig>  distributed genes shared among some strains ,  <dig>  unique genes , and  <dig>  core gene families, of which  <dig>  genes are present in a single copy in all strains and  <dig> gene families are present in multiple copies.
the number of core genes  converged to  <dig>  at the 22nd genome, while the number of pan genes reached  <dig>  genes at the 22nd genome .
the extrapolated number of core genes is very close to the number of core genes  leavis et al.
reported in their microarray-based study which used  <dig> isolates, yet the estimated number of pan genes is higher in the present analysis  <cit> .
furthermore, this study differs slightly from the analysis of van schaik et al.
which estimates the e. faecium core genome to be 2172 ±  <dig> cds  <cit> .
our data do, however, concur with the conclusion that a sizeable fraction of the e. faecium genome is accessory and that the pan genome is considered to open.
phylogenetic, multi-locus sequence typing  and gene content similarity analysis
analysis of the  <dig> e. faecium genomes  showed that the isolates separate into two clades, one branch consisting mostly of ca isolates, with most ha isolates found in the other, as was noted in our previous study  <cit>  .
when analyzing the phylogenetic distances among these  <dig> isolates using  <dig> single-copy ortholog genes of the same length , similar clade patterns were observed for the e. faecium strains as seen in the  <dig> core gene analysis by galloway-pena et.al  <cit> .
all isolates predicted to be part of the cc <dig> genogroup  <cit>  cluster more closely together and branched more distantly than other ha-clade isolates .
the dendogram construction from the gene content dissimilarity represented by jaccard distance  also showed most hospital-isolated strains cluster together except hospital- isolated strain  <dig> , <dig> which was shown genetically to belong to the ca clade.
in addition, although e <dig> is a community- isolated fecal strain, it is genetically closer to the ha strains.
the phylogenetic and gene content dissimilarity analysis results all support the existence of two very distinct clades of e. faecium, which has been previously described using pyrosequencing, microarray, and the concatenation of a  <dig> core genes, estimated to have diverged anywhere from  <dig>  to  <dig> million years ago  <cit> .
ahybrid genome with ~1/ <dig> of the core genes from the ca clade and 2/ <dig> from the ha clade.
bindicates this information was not available.
ca rifampin- and fusidic acid-resistant derivative of clinical strain e. faecium d344s in which the spontaneous loss of pbp <dig> and its surrounding region resulted in an ampicillin-susceptible phenotype.
comparison of e. faecium tx16’s predicted proteins to predicted proteins from the other  <dig> e. faecium genomes using blastp revealed a mosaic-like structure, as previously described  <cit> , and many highly variable regions.
some of the tx <dig> variable regions are ha clade specific .
notably, regions from  <dig> to 38 kb, from  <dig> to 606 kb, from  <dig> to 717 kb, from  <dig> to  <dig>  kb, from  <dig>  to  <dig>  kb and from  <dig>  to  <dig>  kb on the tx <dig> genome are missing or have low identity in the ca strains.
interestingly, region  <dig> to 1802 kb encodes  <dig> surface proteins .
another notable region with low orf identity hits or missing in strain d344srf and tc <dig> is a ~145-kb region from  <dig>  to  <dig>  kb on the tx <dig> genome.
containing the pilus subunit protein ebpcfm  and other  <dig> pilus subunit proteins .
assessment of genomic rearrangements among e. faecium strains was more difficult because other genomes are not complete.
we further investigated the genes that are unique to the ha-clade based on clade assignment of the strains in the phylogenetic analysis, and identified  <dig> orfs  that are unique to the ha clade  .
of the  <dig> orfs,  <dig> orfs are conserved in at least half of the ha clade strains including  <dig> orfs which are shared among all ha-clade isolates.
most of the ha clade unique genes are transposon-related genes, transporters, and prophage genes.
interestingly, a cna b-type gene, the enterocin a operon, and two fms genes  are among the ha-clade specific genes.
strain  <dig> , <dig> was excluded from the ha unique gene analysis because it was previously shown to be a hybrid strain that contained both ha  and ca  alleles based on our  <dig> core gene analysis  <cit> .
mobile genetic elements
e. faecium isolates from patients typically have many mobile genetic elements which often contain antibiotic resistance genes that are easily transferable between strains.
bacteriophage-mediated transduction can transfer antibiotic resistance between enterococci  <cit>  and many bacteriophages have also been identified in e. faecium <cit> .
to identify phage genes on the tx <dig> genome, prophinder and prophage finder were used to search for prophage loci  <cit> .
both programs identified that two chromosomal regions  with a total size of about 62 kb contain phage-related genes.
sixty-one and twenty one phage-related genes were identified in these regions, respectively .
all ca strains have low identity orf hits or missing orfs in the predicted prophage locus from  <dig> to 857 kb, while most ha strains have similar orfs in this locus.
all ca strains and most ha strains lack similar orfs in the other predicted prophage locus from  <dig>  to  <dig>  kb .
in addition to these two main regions, small numbers of phage-related genes were also identified throughout the chromosome, but these were not further analyzed.
is elements and transposases are major mobile genetic elements in e. faecium and about  <dig> is element and transposase-related genes were identified in the tx <dig> genome .
about half of these is elements and transposases are present on the three plasmids.
considering the sizes of the chromosome and three plasmids , plasmid dnas appear to be more susceptible to is element/transposase insertions.
some is elements/transposases exist as multiple copies in specific locations on the chromosome or plasmids.
four copies of isenfa <dig> sequence  were identified in the chromosome but not in the  <dig> tx <dig> plasmids whereas the sequences of is <dig> , is <dig> , and isefa <dig>  were identified in the three plasmids but not in the chromosome.
is elements and transposases were found more frequently in ha strains than in ca strains.
previously, is <dig> was suggested as a molecular screening marker to predict e. faecium pathogenicity because of its presence in clinical e. faecium isolates  <cit> .
we performed a blast search of the  <dig> e. faecium genomes to identify the is/transposase elements showing the same presence or absence patterns of is <dig> .
many is/transposase elements were found to have the same pattern of presence/absence in different strains as is16; including isenfa <dig> , is116/is110/is <dig> family transposases , is <dig> transposases , and transposases .
therefore, all these is elements and transposases  have potential as molecular markers to identify clinical e. faecium.
however, these is elements and transposases are not found in all ha-clade strains as  <dig> ,501; e1039; and e <dig> do not have these is elements and transposases, although they are present in all of the isolates considered to be part of the cc <dig> genogroup .
genomic islands
a pathogenicity island containing the esp gene has previously been reported in e. faecium <cit> .
the esp gene is not present in the tx <dig> genome but a search for other possible genomic islands  in tx <dig> using gi prediction programs including islandpath-dimob  <cit> , sigi-hmm  <cit> , and islandpick  <cit> , identified a total of  <dig> regions totaling  <dig>  bp predicted as gis.
the gis are shown in figure  <dig>  and the genes encoded by gis are listed in additional file 4: table s <dig> and additional file 6: table s <dig>  gis  <dig>   <dig> and  <dig> might be a single gi, since they are located very close together.
gis  <dig> and  <dig> are separated by only  <dig> orfs and  <dig> orfs are present between gis  <dig> and  <dig>  the  <dig> predicted gis have hypothetical proteins and transposon-related proteins in common.
among these putative gis, islands  <dig>   <dig>   <dig>  and  <dig> were frequently present in e. faecium of ha origin .
island  <dig> contains  <dig> genes .
island  <dig> contains  <dig> genes including  <dig> hypothetical proteins,  <dig> predicted abc transport genes, a transposase, a mg-dependent dnase, a lysm family protein, a cell wall protein, and a predicted fosfomycin resistance protein.
island  <dig> and  <dig> are composed of  <dig> and  <dig> genes, respectively.
island  <dig> contains  <dig> hypothetical proteins, a putative membrane protein, and a putative transposase.
four hypothetical proteins and  <dig> transposase related proteins were present in island  <dig>  the presence of a transposase in each island supports that these islands were acquired through horizontal gene transfer.
while a potential role in pathogenesis has been suggested, there are many hypothetical proteins in each island and no genetic or experimental evidence to indicate such a role.
however, island  <dig> which contains a predicted fosfomycin resistance protein might be important in promoting e. faecium colonization because of the selective advantage conferred when this antibiotic is used.
the remaining gis  <dig>   <dig>   <dig>   <dig>  and  <dig> exist only in the tx <dig> genome or in a limited number of e. faecium strains.
we also searched for previously reported gis  <cit>  and pathogenicity islands  <cit>  in the  <dig> e. faecium genomes.
as reported  <cit> , a pathogenicity island including the esp gene was observed in e1162; e1679; and u <dig>  in addition to these three strains, an island with a partial esp gene was also found in  <dig> ,502; c68;  <dig> ,410; tx0133a; and  <dig> , <dig> strains when we performed a blast search.
the esp gene could possibly be intact in these strains but interrupted in the draft assemblies, possibly as a consequence of the next-generation sequencing technology problems.
a gi previously found to be specific to cc <dig>  <cit>  was also observed in the ha clade strains tx0133a; tx82; c68;  <dig> ,410;  <dig> ,933; e1162; tx16;  <dig> ,502; u0317; and e <dig>  intrestingly,  <dig> , <dig>  which is the mosaic strain  <cit> , lacked this gi.
the presence of a putative three-gene pilus-encoding cluster, fms11-fms19-fms <dig>  previously proposed as a small gi  <cit> , is described within the subsequent section on mscramm-like proteins.
genetic loci in e. faecium tx <dig> predicted to be involved in biosynthesis of surface polysaccharides
our analysis of the e. faecium tx <dig> genome did not identify close homologs of the cpsc-k cluster of e. faecalis.
homologs of the two genes, cpsa and cpsb, were found and well conserved in tx <dig>  but were recently reported to not be sufficient for capsule production in e. faecalis <cit> .
similarly, homologs of cpsa-cpsb but not of cpsc-k were found in the  <dig> other e. faecium draft genomes.
in contrast, a locus homologous to the epa locus, which was shown to produce a rhamnose, glucose, galactose, n-acetylgalactosamine and n-acetylglucosamine-containing antigenic cell wall polysaccharide in e. faecalis og1rf <cit> , was found in the tx <dig> genome .
however, identities of the encoded epa-like proteins vary widely between orthologs of tx <dig> and og1rf  to 92% ).
in addition, gene composition and order of the epa-like locus are partially different in these two organisms; the homologs of the three genes in the middle of the e. faecalis epa cluster, epai, epaj and epak, are not present in tx <dig>  while two other epa-like genes, epap and epaq are located at this site.
all  <dig> epa-like genes of tx <dig> were found to be present, highly conserved and similarly organized in all  <dig> available e. faecium draft genomes , indicating that they are part of the core genome of this species.
however, the absence of three epa genes in e. faecium, one encoding a glycosyl hydrolase , suggests the epa polysaccharides of the two species have different sugar compositions.
genes encoding proteins predicted to be an initiating transferase of polysaccharide biosynthesis , glycosyl transferases, acetyl transferases, sugar phosphate transferases and repeat unit polymerases are typically clustered together in loci that mediate polysaccharide synthesis in gram-positive bacteria.
our search for these features in the tx <dig> genome identified two additional regions that might be involved in polysaccharide production.
the first of these regions found in tx <dig>  is a downstream extension of the epa-like region , immediately preceded by an undecaprenyl-phosphate galactose-phosphotransferase  .
unlike the epa region, however, the extension  is present in only  <dig> of the other e. faecium draft genomes; all except one of these strains  belong to the ha clade .
this locus was also observed in these strains by palmer et al.
<cit> .
tx <dig> and these  <dig> draft genomes also have an additional orf , encoding a putative member of the large beta-lactamase-like superfamily  between epao and epar on the upstream side of this region  and a transposase  in  <dig> of the  <dig> genomes on its downstream side.
analysis of the remaining  <dig> draft genomes for a corresponding region revealed a predicted polysaccharide-encoding gene cluster downstream of the epa region in all of them, , although these regions have only low similarities to those of tx <dig> and the  <dig> genomes above and extensive sequence variation among each other .
locus  <dig>  was found in only ha clade strains, while locus  <dig>  and locus  <dig> , although found in some ha-clade strains, were only found in non-cc <dig> isolates as well as in four of the five ca-clade isolates, indicating some specificity of polysaccharide biosynthesis genes for certain lineages or niches.
of note, none of locus  <dig> strains have is <dig>  only two of the locus  <dig> strains have is <dig>  while all that had locus  <dig> or  <dig> have is <dig>
the second region found in tx <dig> that appears likely to be involved in polysaccharide biosynthesis  is largely unique to this genome, with only the first four orfs present in  <dig> of the genomes and the whole region completely absent in one of the genomes .
however, each of the  <dig> other genomes has additional genes predicted to be involved in polysaccharide biosynthesis at this location.
distribution of genes encoding mscramm-like proteins, putative virulence genes, antibiotic resistance determinants, and crisprs
previous studies of e. faecium tx <dig> identified  <dig> genes encoding lpxtg family cell-wall anchored proteins with mscramm-like features, such as immunoglobulin-like folding;  <dig> of these were found in four gene clusters, each predicted/demonstrated to encode a different pilus, and four were found as individual mscramm-encoding genes  <cit> .
our search for these genes in  <dig> unique e. faecium draft genomes in this study found all of the mscramm-encoding genes to be widely distributed except fms <dig>  and fms <dig> which were only in ha-clade isolates  .
moreover, our analysis revealed that ebpa-ebpb-ebpcfmfms14-fms17-fms13fms <dig>  scm, and fms <dig>  all have sequence variants in some of the  <dig> strains, with identities of the encoded variant proteins ranging from 39%  to 94%  versus their counterparts in tx <dig> .
in general, most of the mscramms followed the ca/ha clade groupings with a variant representing each clade.
variant  <dig> of the fms11-fms19-fms <dig> locus was strictly found in the ha-clade, and variant  <dig> in the ca-clade except for  <dig> , <dig> which only had one of the three proteins  as a ca-variant, suggesting recombination by this isolate.
variant  <dig> of fms14-fms17-fms <dig> was found in all but one ha clade isolate  and variant  <dig> in all  <dig> ca-clade strains.
variant  <dig> of scm was found to be exclusively carried by all  <dig> ha clade strains and variant  <dig> by  <dig> of the  <dig> ca clade strains.
although the differences between these mscramms in ca- vs. ha-clade strains are generally greater  than the differences  previously reported for the clade-specific differences in a set of core genes that excluded predicted surface proteins, they are comparable to the differences seen in several other surface proteins that have been studied  <cit> .
interestingly, the majority of ha clade strains  were found to have variant  <dig> of the ebp pilus operon, while variant  <dig> was exclusively found in the  <dig> ca-clade strains in addition to variant  <dig> in three of the five isolates.
in contrast, variation within fms <dig> was restricted to the ha clade; all ca clade isolates carried fms <dig> variant  <dig>  but the percent identity between these two variants is much smaller , possibly indicating the need for a new gene name.
also of note was the acm gene, which is present as a pseudogene in all of the ca-clade isolates except  <dig> , <dig> which is the only ca-clade isolate that is from a hospitalized patient; acm pseudogenes were also found in non-cc <dig> ha-clade isolates.
of note, our search for mscramms and potential pilus proteins also found one to three new individually encoded cnab domain-containing mscramms in five of the e. faecium draft genomes and a new pilus encoding gene cluster in strain e1071; the latter consists of three genes one of which is a relatively distant homolog of bee <dig>  and two are identical or highly homologous to bee <dig> or bee <dig>  of a plasmid-encoded bee pilus gene cluster found in a small percentage of e. faecalis isolates  <cit> .
to identify possible virulence genes in the e. faecium genomes, the enterococcal virulence factors listed in the virulence factors database   <cit>  were aligned to the orf protein sequences using blastp and filtered with 50% identity and 50% match length.
the homologs of efaa, ef <dig> , cpsa and cpsb genes are present in all e. faecium strains , and espefm and hylefm are exclusively present in some ha clade strains while the homolog of ef <dig>  is exclusively present in the ca-clade strains .
homologs of other e. faecalis virulence factors listed in the vfdb were not found in tx <dig> genome.
we also searched the  <dig> e. faecium isolates for the presence and absence of  <dig> resistance genes.
our data correspond to previously published data for some of the isolates  <cit> .
we observed that there is a clear distinction between the isolates of the genetically defined ca clade and those of the ha clade with none of the ca clade isolates having any of the antibiotic resistance determinants analyzed .
on the other hand, all of the ha-clade isolates have multiple resistance determinants, including the pbp5-r allele that confers ampicillin resistance previously reported by galloway-pena et al.
<cit> , except for strains  <dig> , <dig> and e <dig>   <dig> , <dig>  which is in the ha-clade but lacks all antibiotic resistances including pbp5-r, may have lost the allele via recombination and acquired pbp5-s or may even represent a more ancestral isolate.
indeed,  <dig> , <dig> was shown to be a hybrid of ha and ca genomes by palmer, et al., with the replacement  region including pbp5-s, which could explain the origin of pbp5-s in this strain  <cit> .
e <dig>  which has the pbp5-r allele but none of the other resistance genes, is genetically defined as a ha-clade isolate, but came from a healthy volunteer, perhaps explaining its lack of other antibiotic resistances.
interestingly, neither of these strains has is <dig>  d344srf is the only other ha-clade isolate that lacks the pbp5-r allele; however, this strain is known to have spontaneously lost pbp <dig> and the surrounding region and contains many other resistances  <cit> .
of note, e <dig> only has two of the  <dig> resistances analyzed ; however, this could possibly be explained by its early isolation in  <dig>  this again suggests that these isolates are more distantly related to the other strains within the ha-clade.
aa rifampin- and fusidic acid-resistant derivative of clinical strain e. faecium d344s in which the spontaneous loss of pbp <dig> and its surrounding region resulted in an ampicillin-susceptible phenotype.
bamino acid change  in residue  <dig> or  in residue  <dig> of gyra.
camino acid change  in residue  <dig> or  in residue  <dig> of parc.
dconsensus sequence of the pbp5-r allele encoding the low affinity pbp5-r.
etc <dig> was not included in this analysis as it is a transconjugant of c <dig> and d344srf, so therefore is not a unique genome.
two groups have previously analyzed crispr-associated genes within e. faecalis and e. faecium genomes  <cit> .
partial crispr-like loci were previously described in e <dig>  e <dig>  and u0317; however, these loci were within a gene and were considered non-functional  <cit> .
in addition, palmer et al.
identified crispr-cas predicted proteins in the broad institute strains com12;  <dig> ,733; and  <dig> , <dig>  <cit> .
similarly, we only found a crispr-cas locus in strain tx <dig>  out of the  <dig> strains not previously studied .
in summary, out of the  <dig> available genomes, only one of the ha-clade isolates contained crisp-loci, namely the hybrid strain  <dig> , <dig>  the three other strains containing crispr-loci of the ca-clade  all lacked antibiotic resistance determinants.
therefore, our data coincide with the previous observation that members of the recently emerged high-risk enterococcal lineages lack crispr-loci and the inverse relationship between the presence of a crispr-cas locus and acquired antibiotic resistance  <cit> .
metabolic pathway
metabolic pathways of e. faecium might have contributed to the recently increased incidence of e. faecium colonization and infection.
to help understand e. faecium metabolism, the kegg pathway  and kaas  databases were used.
both databases predicted more than  <dig> pathways using tx <dig> genomic information.
e. faecium exhibits major genomic differences in the genes involved in energy metabolism compared to that of other facultative anaerobic bacteria.
however, like other species in the lactobacillaceae order, genes for typical aerobic energy  generation through the tca cycle and electron transport chain do not exist, i.e., genes encoding complex i , ii , iii , and iv .
when we compared the metabolic pathways of tx <dig> to those of e. faecalis v <dig> using the kegg database, all  <dig> metabolic pathways of e. faecalis were also predicted in tx <dig>  indeed, more diverse metabolic activities were observed in tx <dig> .
additional files 10: table s <dig> and additional files 11: table s <dig> show lists of enzymes that only exist in e. faecium tx <dig> or e. faecalis v <dig> when kegg enzymes from both strains were compared.
many of these enzymes were also described by van schaik et al.
who compared  <dig> european strains  to e. faecalis v <dig>  they found  <dig> cogs present in their e. faecium genomes lacking in v <dig>  whereas we found  <dig> predicted enzymes present in tx <dig> lacking in e. faecalis v <dig> according to kegg analysis.
additionally, they found  <dig> cogs specific for e. faecalis v <dig>  compared to the european strains, whereas we found only  <dig> enzymes specific to v <dig> when compared to tx <dig> according to kegg analysis  <cit> .
plasmids
alignment of orfs from the three plasmids of tx <dig> to the orfs from the other  <dig> e. faecium genomes by blastp showed that all strains shared some orfs that are similar to the orfs of the three e. faecium tx <dig> plasmids , but none of them have more than 90% of the orfs from any of the plasmids.
it is likely that some strains may have similar but not identical plasmids as tx <dig>  but identification of plasmids in other strains is difficult since those genomes are draft sequences.
alignment of orfs of the three tx <dig> plasmids to  <dig> complete e. faecium plasmid sequences available in ncbi using tblastn with 90% identity and 50% match length cutoffs showed that pdo <dig> is most similar to plasmid pm7m <dig>  a  <dig>  kb plasmid which shared  <dig> orfs of the  <dig> orfs  from pdo <dig>  and that pdo <dig> is somewhat similar to plasmids prum and ps <dig> with  <dig> % and  <dig> % match to pdo <dig> orfs respectively.
tx <dig> plasmid pdo <dig> does not seem to be similar to any completely sequenced e. faecium plasmids but has similarity to the partially sequenced e. faecium large plasmid plg <dig>  both pdo <dig> and plg1plasmids harbor the hyaluronidase gene , the hylefm gene was also found in ha strains  <dig> , <dig>   <dig> , <dig>   <dig> , <dig>  c <dig>  tc <dig> and u <dig>
discussion
tx <dig> was the first e. faecium strain sequenced and has been used in various studies since  <cit> .
the tx <dig> genome is characterized by numerous hyper variant loci and a large number of is elements and transposons.
ortholog analysis as well as core and pan-genome analysis of tx <dig> and the other  <dig> sequenced strains revealed that e. faecium genomes are highly heterogeneous in gene content and possess a large number of dispensable genes.
similar to the findings by van schaik et al.
<cit> , pan and core genome analysis predict the pan genome to be open.
phylogenetic analysis using single-copy orthologs of the same length and gene content dissimilarity analysis in addition to recent studies  <cit>  looking at core genes, snps and 16s rrna, all indicate a large divergence between ca-clade isolates and ha-clade isolates.
furthermore, our previous analysis  <cit>  and analyses within this study show that cc <dig> genogroup isolates cluster more closely together and further away from the ca-clade isolates than the other non-cc <dig> ha-clade isolates, indicating the cc <dig> genogroup is a more recently evolved genogroup.
genomic island analysis by codon usage bias and composition variation showed that tx <dig> has  <dig> gis, although tx <dig> also possesses a large number of hyper variant loci, suggesting that most of the genomic variable loci in tx <dig> were acquired through lateral gene transfer, possibly through mobile elements such as transposons.
in general, strains in the ha clade harbored more transposons than the ca strains and certain is elements such as is <dig>  these findings are consistent with a previous study using whole genome microarray  <cit> .
although is <dig> presence has been proposed as an indicator of hospital-associated strains such as those apart of the cc <dig> genogroup  <cit> , is <dig> was not found in all ha-clade strains.
of note, however, all ha-clade strains contained the pbp5-r allele  which may indicate that this is a reliable marker for hospital-associated isolates.
indeed, the pbp5-r allele is also found in animal and community isolates that are considered within the ha-clade, but not considered clinically associated  <cit> .
the exception,  <dig> , <dig> is interesting in that it is the ha-clade isolate from the blood of a hospitalized patient with no resistance genes, possibly supporting the concept that the genomic content of a strain, not just antibiotic resistance, adds to the survival in the hospital environment.
in the  <dig> gene analysis by galloway-pena et al., it was found that  <dig> of the  <dig> genes of this strain studied grouped with the community clade, indicating it is a hybrid strain  <cit>  as also reported in a recent study  <cit> .
capsular and other cell envelope polysaccharides of several gram-positive bacteria are known to have important roles in virulence and protective immunity  <cit> .
although the majority of studies on enterococcal surface polysaccharides have focused on e. faecalis, similar molecules have also been identified in e. faecium and suggested as targets for opsonic antibodies and as potential vaccine candidates  <cit> , and also implicated in resistance of tx <dig> to phagocytosis in normal human serum  <cit> .
two such gene clusters, cps and epa, have been identified in e. faecalis <cit> .
although a 7-9-gene cps region  was recently determined necessary for the production of an e. faecalis capsular polysaccharide  <cit>  and shown to contribute to pathogenesis and evasion of the host innate immune response  <cit> , tx <dig> only contains two homologs of the genes in this locus  <cit> .
in contrast,  <dig> of the  <dig> e. faecalis epa polysaccharide genes have homologs in tx <dig> and the other  <dig> e. faecium genomes, although their sequences vary between the two species.
therefore, it is likely that e. faecalis and e. faecium produce compositionally related, but not identical, epa surface polysaccharides.
the hyper variable nature of the two polysaccharide loci found in tx <dig> raises the possibility that they are involved in biosynthesis of antigenically diverse surface polysaccharides which could help protect e. faecium against host immune responses.
similar to other gram-positive bacteria, various mscramm-like cell wall anchored proteins have been previously identified in e. faecium; these include the collagen adhesin acm and biofilm-associated ebp pili, shown to be important for endocarditis and uti in animal models  <cit> , respectively, as well as two other collagen-binding mscramms, scm and fms <dig>   <cit> .
our comparison of  <dig> previously described mscramm and pilus encoding genes of tx <dig>  <cit>  with those of  <dig> e. faecium draft genomes found them to be common among these strains and the majority of them  to be enriched among ha clade strains or have a sequence variant mostly/exclusively carried by ca clade strains.
thus, these findings agree with previous hybridization results  <cit>  and with the presence of two distinct subpopulations of e. faecium.
furthermore, one of these genes, acm, was previously found to be expressed more often by clinical versus non-clinical isolates, whereas a pseudogene was often found in isolates from the community  <cit> .
taken together, these data indicate a clear difference in the mscramm and pilus gene profiles of the ha and ca clades, suggesting that these genes may have favored the emergence of ha-clade e. faecium in nosocomial infections.
when we combined our finding with previously published results, four of the  <dig> e. faecium genomes contain the crispr-cas locus.
three of these strains are within the ca clade and lack all antibiotic resistances analyzed in this study.
one of the strains,  <dig> , <dig>  is a unique strain in which its genome is a hybrid of ca and ha genes.
however, it does have  <dig> antibiotic resistance associated genes, showing there is not always an inverse relation between the number of antibiotic resistance determinants and the presence of crispr loci.
more strains containing crispr-loci will need to be studied in order to determine if  <dig> , <dig> is just an exception to the rule, or if the highly recombinant nature of e. faecium makes it different from e. faecalis with respect to the presence of crispr-loci in relation to antibiotic resistance determinants.
overall, there seem to be some patterns that point to specific evolutionary events throughout e. faecium’s history as a species.
first and foremost, there is a large ancestral split between the ca- and ha-clade strains which are separated by at least a 3–4% difference in their core genome  <cit> .
the ca-clade isolates, except one, do not have either polysaccharide synthesis locus  <dig> or  <dig> downstream of the epa region, antibiotic resistance genes, certain genomic islands, or is elements.
after the ha-clade diverged from ca-clade there was further evolution within the ha clade and some ha-clade strains studied here may represent phylogenetic transitional lineages .
like the ca-clade strains, these transitional lineages are characterized by a lack of is <dig>  and have neither locus  <dig> nor  <dig>  in the epa extension.
although the data are limited, one scenario that could explain these observations is if locus  <dig> replaced locus  <dig> in a ha-clade ancestral strain, after the split from the ca clade, which later acquired is <dig> and then, subsequently, locus  <dig> or  <dig> replaced locus  <dig> in the epa extension region.
even if this is not the case, it seems clear that only strains further along in the phylogenetic trees, indicating a division within the ha-clade , acquired is <dig> and the polysaccharide biosynthesis loci  <dig> and  <dig>  the exception is e <dig>  a strain previously shown to have  <dig> of  <dig> genes from the ha-clade, which could have gained locus  <dig> via recombination.
also of note, three of the four strains that have locus  <dig> downstream of the epa locus lack the ebp genes, possibly suggesting there may have been some kind of gain and loss through homologous recombination.
figure  <dig> shows the projected scenarios for the evolution of the two clades of e. faecium as can be envisioned using our data as well as other previous publications  <cit> .
the hypothesis is that there was a primordial type of e. faecium which split many millinea ago and evolved into two early community groups which had homologous genes e.g.
the pbp5-s or pbp5-r alleles, the latter representing community sources of are .
these lineages could recombine with each other resulting in hybrid strains  .
the divergence between the two community groups eventually reached a core genomic difference of approximately 3–4%, creating a ha clade, which includes both ampicillin- resistant, community-based isolates, such as those from some canine and feline origins, as well as most of the clinical-, hospital- and outbreak- associated isolates and a ca clade, which consists mostly of community derived isolates.
most likely, community and hospital are isolates split from the same ancestor, as represented by scenario two.
however, it is also possible that are clones evolved from the animal reservoir , or that animal are isolates represent evolutionary descendants of hospital are transferred from humans to their pets .
CONCLUSIONS
in conclusion, the completion of the tx <dig> genome has provided insight into the intricate genomic features of e. faecium, and will surely serve as an important reference for those studying e. faecium genomics in the future.
by studying tx <dig>  an endocarditis isolate belonging to cc <dig>  and comparing the tx <dig> genome to the other  <dig> draft genomes, we have been able to confirm the high genomic plasticity of this organism.
the ha-clade isolates contain a number of unique is elements, transposons, phages, plasmids, genomic islands, and inherent and acquired antibiotic resistance determinants, most likely contributing to the emergence of this organism in the hospital environment that has occurred in the last 30 years.

 enterococci are normal constituents of the gastro-intestinal flora of humans and other animals. although they only occasionally cause infections in healthy individuals, they are the third most commonly isolated gram positive organisms from hospital-associated infections in the united states and are increasingly reported in other countries. enterococcal infections are often difficult to treat due to the number of antibiotics to which these organisms are resistant. some antibiotic resistances are intrinsic, such as resistances to cephalosporins, while other antibiotic resistances are acquired through mutations or horizontal gene transfer, most notably the van systems that encode vancomycin resistance. several recent studies also confirmed that enterococci can transfer their resistance to even more virulent organisms, such as staphylococcus aureus. enterococcus faecalis is the most common enterococcal species recovered from infections. however, in the last decade, infections with enterococcus faecium have been on the rise in the united states, europe, and south america. in the us, isolates of e. faecium now account for ca. 35% of nosocomial enterococcal isolates identified to the species level. it is still not clear what has caused the ecological replacement of e. faecalis with e. faecium in the nosocomial setting, but it is speculated that the intense use of antibiotics in hospitals and the multiple antibiotic resistances of e. faecium have been major contributing factors. a few genes have been suggested as being virulence determinants in e. faecium due to their enrichment in clinical isolates, such as the fms or hyl genes. however, only three genes have been experimentally implicated to have an impact on virulence in animal models, namely esp, which has a role in biofilm, urinary tract infection, and endocarditis; acm, encoding a collagen binding adhesin contributing to endocarditis; and the ebpfm operon which encodes pili that are important in biofilm and urinary tract infection. in addition, conjugative transfer of a plasmid with a hyl-like gene not only conferred increased resistance to vancomycin but also increased virulence in transconjugants in the mouse peritonitis model, and a different hyl-plasmid conferred colonization in the murine gut. while the gene responsible for this increase in virulence and colonization have yet to be determined, the deletion of the hyl gene did not cause attenuation in the peritonitis model. molecular epidemiological studies of outbreaks of e. faecium using mlst initially indicated that there was a specific lineage or genogroup of strains, designated clonal complex that was predominant in the hospital environment. other studies using pyrosequencing and whole-genome microarray subsequently indicated that, while there appeared to be a globally dispersed clade containing the vast majority of epidemic and clinical isolates which harbor a large content of accessory genes specific to this clade, isolates associated with healthcare settings were not strictly clonally related to each other. in particular, while cc genogroup isolates are part of the ha subpopulation, not all ha isolates are considered part of the st lineage. recent studies in our laboratory and others have shown large differences in the sequence of the core genome, as well as differences in the 16-s rrna, between two different clades which were named the hospital-associated clade and community-associated clade strains,. the ha clade contains most clinical and ha-associated strains but also included strains from non-hospital origin. molecular studies and comprehensive comparative genomic studies of e. faecium have long been hindered by the lack of a complete genome sequence. the tx genome was initially sequenced at the department of energy’s joint genome institute in walnut creek, ca. in in an effort to demonstrate capabilities of the sequencing technology at that time by sequencing the genome in only 1 day. however, the genome was far from closed and the past decade has been spent on annotation, final assembly, and analyses of this genome. recently, while this manuscript was in review, a closed e. faecium genome was published by lam et al. using the st isolate aus which was isolated from the bloodstream of a patient in melbourne, australia. in this study, we report the closed genome of the us e. faecium endocarditis isolate tx, and a comparative analysis of this strain’s genome with other available e. faecium draft genomes, as well as the recently published aus. due to the fact the tx genome has been used in multiple pathogenesis studies and is a part of the clonal group representing the majority of clinical strains globally, the complete genome sequence of e. faecium tx will facilitate future research by providing a critical starting point for genome-wide functional studies to determine the molecular basis of pathogenesis and to further understand the evolution and molecular epidemiology of e. faecium infective strains.  e. faecium tx general genome features the e. faecium tx genome consists of one chromosome and three plasmids. the chromosome contains,137 bp with protein-coding orfs, trnas, copies of ribosomal rrna and other non-coding rnas. the chromosome has a gc content of %, and it shows a clear gc skew at the origin of replication. the sizes of the three plasmids are, and  bp, encoding and orfs, respectively. to investigate the conservation of the gene order of e. faecium compared to its close relative e. faecalis, a blastp alignment of all the predicted proteins from the tx and v genomes was performed followed by orf synteny analysis using dagchainer. the result showed that e. faecium tx gene order is very different from that of e. faecalis strain v and all orf synteny blocks were relatively short. interestingly, when comparing tx to the closed genome aus which was published while this paper was in review, mauve genome alignment analysis resulted in locally collinear blocks for both tx and aus ranging from –  bp for tx and –  bp for aus. the two isolates had very similar synteny, although two regions found in tx were inverted in aus two site-specific tyrosine family recombinases were found flanking these two inversions. the genome size of the e. faecium strains vary substantially from  mb to  mb, while the number of orfs varies from to. ortholog analysis of tx compared to tx and all the available but unfinished e. faecium genomes using blastp of predicted protein sequences and orthomcl resulted in distributed genes shared among some strains, unique genes, and core gene families, of which genes are present in a single copy in all strains and gene families are present in multiple copies. the number of core genes converged to at the 22nd genome, while the number of pan genes reached genes at the 22nd genome. the extrapolated number of core genes is very close to the number of core genes leavis et al. reported in their microarray-based study which used isolates, yet the estimated number of pan genes is higher in the present analysis. furthermore, this study differs slightly from the analysis of van schaik et al. which estimates the e. faecium core genome to be 2172 ±  cds. our data do, however, concur with the conclusion that a sizeable fraction of the e. faecium genome is accessory and that the pan genome is considered to open. phylogenetic, multi-locus sequence typing and gene content similarity analysis analysis of the e. faecium genomes showed that the isolates separate into two clades, one branch consisting mostly of ca isolates, with most ha isolates found in the other, as was noted in our previous study. when analyzing the phylogenetic distances among these isolates using single-copy ortholog genes of the same length, similar clade patterns were observed for the e. faecium strains as seen in the core gene analysis by galloway-pena et.al. all isolates predicted to be part of the cc genogroup cluster more closely together and branched more distantly than other ha-clade isolates. the dendogram construction from the gene content dissimilarity represented by jaccard distance also showed most hospital-isolated strains cluster together except hospital- isolated strain, which was shown genetically to belong to the ca clade. in addition, although e is a community- isolated fecal strain, it is genetically closer to the ha strains. the phylogenetic and gene content dissimilarity analysis results all support the existence of two very distinct clades of e. faecium, which has been previously described using pyrosequencing, microarray, and the concatenation of a core genes, estimated to have diverged anywhere from to million years ago. ahybrid genome with ~1/ of the core genes from the ca clade and 2/ from the ha clade. bindicates this information was not available. ca rifampin- and fusidic acid-resistant derivative of clinical strain e. faecium d344s in which the spontaneous loss of pbp and its surrounding region resulted in an ampicillin-susceptible phenotype. comparison of e. faecium tx16’s predicted proteins to predicted proteins from the other e. faecium genomes using blastp revealed a mosaic-like structure, as previously described, and many highly variable regions. some of the tx variable regions are ha clade specific. notably, regions from to 38 kb, from to 606 kb, from to 717 kb, from to  kb, from to  kb and from to  kb on the tx genome are missing or have low identity in the ca strains. interestingly, region to 1802 kb encodes surface proteins. another notable region with low orf identity hits or missing in strain d344srf and tc is a ~145-kb region from to  kb on the tx genome. containing the pilus subunit protein ebpcfm and other pilus subunit proteins. assessment of genomic rearrangements among e. faecium strains was more difficult because other genomes are not complete. we further investigated the genes that are unique to the ha-clade based on clade assignment of the strains in the phylogenetic analysis, and identified orfs that are unique to the ha clade. of the orfs, orfs are conserved in at least half of the ha clade strains including orfs which are shared among all ha-clade isolates. most of the ha clade unique genes are transposon-related genes, transporters, and prophage genes. interestingly, a cna b-type gene, the enterocin a operon, and two fms genes are among the ha-clade specific genes. strain, was excluded from the ha unique gene analysis because it was previously shown to be a hybrid strain that contained both ha and ca alleles based on our core gene analysis. mobile genetic elements e. faecium isolates from patients typically have many mobile genetic elements which often contain antibiotic resistance genes that are easily transferable between strains. bacteriophage-mediated transduction can transfer antibiotic resistance between enterococci and many bacteriophages have also been identified in e. faecium. to identify phage genes on the tx genome, prophinder and prophage finder were used to search for prophage loci. both programs identified that two chromosomal regions with a total size of about 62 kb contain phage-related genes. sixty-one and twenty one phage-related genes were identified in these regions, respectively. all ca strains have low identity orf hits or missing orfs in the predicted prophage locus from to 857 kb, while most ha strains have similar orfs in this locus. all ca strains and most ha strains lack similar orfs in the other predicted prophage locus from to  kb. in addition to these two main regions, small numbers of phage-related genes were also identified throughout the chromosome, but these were not further analyzed. is elements and transposases are major mobile genetic elements in e. faecium and about is element and transposase-related genes were identified in the tx genome. about half of these is elements and transposases are present on the three plasmids. considering the sizes of the chromosome and three plasmids, plasmid dnas appear to be more susceptible to is element/transposase insertions. some is elements/transposases exist as multiple copies in specific locations on the chromosome or plasmids. four copies of isenfa sequence were identified in the chromosome but not in the tx plasmids whereas the sequences of is, is, and isefa were identified in the three plasmids but not in the chromosome. is elements and transposases were found more frequently in ha strains than in ca strains. previously, is was suggested as a molecular screening marker to predict e. faecium pathogenicity because of its presence in clinical e. faecium isolates. we performed a blast search of the e. faecium genomes to identify the is/transposase elements showing the same presence or absence patterns of is. many is/transposase elements were found to have the same pattern of presence/absence in different strains as is16; including isenfa, is116/is110/is family transposases, is transposases, and transposases. therefore, all these is elements and transposases have potential as molecular markers to identify clinical e. faecium. however, these is elements and transposases are not found in all ha-clade strains as,501; e1039; and e do not have these is elements and transposases, although they are present in all of the isolates considered to be part of the cc genogroup. genomic islands a pathogenicity island containing the esp gene has previously been reported in e. faecium. the esp gene is not present in the tx genome but a search for other possible genomic islands in tx using gi prediction programs including islandpath-dimob, sigi-hmm, and islandpick, identified a total of regions totaling  bp predicted as gis. the gis are shown in figure  and the genes encoded by gis are listed in additional file 4: table s and additional file 6: table s gis and might be a single gi, since they are located very close together. gis and are separated by only orfs and orfs are present between gis and the predicted gis have hypothetical proteins and transposon-related proteins in common. among these putative gis, islands and were frequently present in e. faecium of ha origin. island contains genes. island contains genes including hypothetical proteins, predicted abc transport genes, a transposase, a mg-dependent dnase, a lysm family protein, a cell wall protein, and a predicted fosfomycin resistance protein. island and are composed of and genes, respectively. island contains hypothetical proteins, a putative membrane protein, and a putative transposase. four hypothetical proteins and transposase related proteins were present in island the presence of a transposase in each island supports that these islands were acquired through horizontal gene transfer. while a potential role in pathogenesis has been suggested, there are many hypothetical proteins in each island and no genetic or experimental evidence to indicate such a role. however, island which contains a predicted fosfomycin resistance protein might be important in promoting e. faecium colonization because of the selective advantage conferred when this antibiotic is used. the remaining gis and exist only in the tx genome or in a limited number of e. faecium strains. we also searched for previously reported gis and pathogenicity islands in the e. faecium genomes. as reported, a pathogenicity island including the esp gene was observed in e1162; e1679; and u in addition to these three strains, an island with a partial esp gene was also found in,502; c68;,410; tx0133a; and, strains when we performed a blast search. the esp gene could possibly be intact in these strains but interrupted in the draft assemblies, possibly as a consequence of the next-generation sequencing technology problems. a gi previously found to be specific to cc was also observed in the ha clade strains tx0133a; tx82; c68;,410;,933; e1162; tx16;,502; u0317; and e intrestingly,, which is the mosaic strain, lacked this gi. the presence of a putative three-gene pilus-encoding cluster, fms11-fms19-fms previously proposed as a small gi, is described within the subsequent section on mscramm-like proteins. genetic loci in e. faecium tx predicted to be involved in biosynthesis of surface polysaccharides our analysis of the e. faecium tx genome did not identify close homologs of the cpsc-k cluster of e. faecalis. homologs of the two genes, cpsa and cpsb, were found and well conserved in tx but were recently reported to not be sufficient for capsule production in e. faecalis. similarly, homologs of cpsa-cpsb but not of cpsc-k were found in the other e. faecium draft genomes. in contrast, a locus homologous to the epa locus, which was shown to produce a rhamnose, glucose, galactose, n-acetylgalactosamine and n-acetylglucosamine-containing antigenic cell wall polysaccharide in e. faecalis og1rf, was found in the tx genome. however, identities of the encoded epa-like proteins vary widely between orthologs of tx and og1rf to 92% ). in addition, gene composition and order of the epa-like locus are partially different in these two organisms; the homologs of the three genes in the middle of the e. faecalis epa cluster, epai, epaj and epak, are not present in tx while two other epa-like genes, epap and epaq are located at this site. all epa-like genes of tx were found to be present, highly conserved and similarly organized in all available e. faecium draft genomes, indicating that they are part of the core genome of this species. however, the absence of three epa genes in e. faecium, one encoding a glycosyl hydrolase, suggests the epa polysaccharides of the two species have different sugar compositions. genes encoding proteins predicted to be an initiating transferase of polysaccharide biosynthesis, glycosyl transferases, acetyl transferases, sugar phosphate transferases and repeat unit polymerases are typically clustered together in loci that mediate polysaccharide synthesis in gram-positive bacteria. our search for these features in the tx genome identified two additional regions that might be involved in polysaccharide production. the first of these regions found in tx is a downstream extension of the epa-like region, immediately preceded by an undecaprenyl-phosphate galactose-phosphotransferase. unlike the epa region, however, the extension is present in only of the other e. faecium draft genomes; all except one of these strains belong to the ha clade. this locus was also observed in these strains by palmer et al.. tx and these draft genomes also have an additional orf, encoding a putative member of the large beta-lactamase-like superfamily between epao and epar on the upstream side of this region and a transposase in of the genomes on its downstream side. analysis of the remaining draft genomes for a corresponding region revealed a predicted polysaccharide-encoding gene cluster downstream of the epa region in all of them,, although these regions have only low similarities to those of tx and the genomes above and extensive sequence variation among each other. locus was found in only ha clade strains, while locus and locus, although found in some ha-clade strains, were only found in non-cc isolates as well as in four of the five ca-clade isolates, indicating some specificity of polysaccharide biosynthesis genes for certain lineages or niches. of note, none of locus strains have is only two of the locus strains have is while all that had locus or have is the second region found in tx that appears likely to be involved in polysaccharide biosynthesis is largely unique to this genome, with only the first four orfs present in of the genomes and the whole region completely absent in one of the genomes. however, each of the other genomes has additional genes predicted to be involved in polysaccharide biosynthesis at this location. distribution of genes encoding mscramm-like proteins, putative virulence genes, antibiotic resistance determinants, and crisprs previous studies of e. faecium tx identified genes encoding lpxtg family cell-wall anchored proteins with mscramm-like features, such as immunoglobulin-like folding; of these were found in four gene clusters, each predicted/demonstrated to encode a different pilus, and four were found as individual mscramm-encoding genes. our search for these genes in unique e. faecium draft genomes in this study found all of the mscramm-encoding genes to be widely distributed except fms and fms which were only in ha-clade isolates. moreover, our analysis revealed that ebpa-ebpb-ebpcfmfms14-fms17-fms13fms scm, and fms all have sequence variants in some of the strains, with identities of the encoded variant proteins ranging from 39% to 94% versus their counterparts in tx. in general, most of the mscramms followed the ca/ha clade groupings with a variant representing each clade. variant of the fms11-fms19-fms locus was strictly found in the ha-clade, and variant in the ca-clade except for, which only had one of the three proteins as a ca-variant, suggesting recombination by this isolate. variant of fms14-fms17-fms was found in all but one ha clade isolate and variant in all ca-clade strains. variant of scm was found to be exclusively carried by all ha clade strains and variant by of the ca clade strains. although the differences between these mscramms in ca- vs. ha-clade strains are generally greater than the differences previously reported for the clade-specific differences in a set of core genes that excluded predicted surface proteins, they are comparable to the differences seen in several other surface proteins that have been studied. interestingly, the majority of ha clade strains were found to have variant of the ebp pilus operon, while variant was exclusively found in the ca-clade strains in addition to variant in three of the five isolates. in contrast, variation within fms was restricted to the ha clade; all ca clade isolates carried fms variant but the percent identity between these two variants is much smaller, possibly indicating the need for a new gene name. also of note was the acm gene, which is present as a pseudogene in all of the ca-clade isolates except, which is the only ca-clade isolate that is from a hospitalized patient; acm pseudogenes were also found in non-cc ha-clade isolates. of note, our search for mscramms and potential pilus proteins also found one to three new individually encoded cnab domain-containing mscramms in five of the e. faecium draft genomes and a new pilus encoding gene cluster in strain e1071; the latter consists of three genes one of which is a relatively distant homolog of bee and two are identical or highly homologous to bee or bee of a plasmid-encoded bee pilus gene cluster found in a small percentage of e. faecalis isolates. to identify possible virulence genes in the e. faecium genomes, the enterococcal virulence factors listed in the virulence factors database were aligned to the orf protein sequences using blastp and filtered with 50% identity and 50% match length. the homologs of efaa, ef, cpsa and cpsb genes are present in all e. faecium strains, and espefm and hylefm are exclusively present in some ha clade strains while the homolog of ef is exclusively present in the ca-clade strains. homologs of other e. faecalis virulence factors listed in the vfdb were not found in tx genome. we also searched the e. faecium isolates for the presence and absence of resistance genes. our data correspond to previously published data for some of the isolates. we observed that there is a clear distinction between the isolates of the genetically defined ca clade and those of the ha clade with none of the ca clade isolates having any of the antibiotic resistance determinants analyzed. on the other hand, all of the ha-clade isolates have multiple resistance determinants, including the pbp5-r allele that confers ampicillin resistance previously reported by galloway-pena et al., except for strains, and e, which is in the ha-clade but lacks all antibiotic resistances including pbp5-r, may have lost the allele via recombination and acquired pbp5-s or may even represent a more ancestral isolate. indeed,, was shown to be a hybrid of ha and ca genomes by palmer, et al., with the replacement region including pbp5-s, which could explain the origin of pbp5-s in this strain. e which has the pbp5-r allele but none of the other resistance genes, is genetically defined as a ha-clade isolate, but came from a healthy volunteer, perhaps explaining its lack of other antibiotic resistances. interestingly, neither of these strains has is d344srf is the only other ha-clade isolate that lacks the pbp5-r allele; however, this strain is known to have spontaneously lost pbp and the surrounding region and contains many other resistances. of note, e only has two of the resistances analyzed; however, this could possibly be explained by its early isolation in this again suggests that these isolates are more distantly related to the other strains within the ha-clade. aa rifampin- and fusidic acid-resistant derivative of clinical strain e. faecium d344s in which the spontaneous loss of pbp and its surrounding region resulted in an ampicillin-susceptible phenotype. bamino acid change in residue or in residue of gyra. camino acid change in residue or in residue of parc. dconsensus sequence of the pbp5-r allele encoding the low affinity pbp5-r. etc was not included in this analysis as it is a transconjugant of c and d344srf, so therefore is not a unique genome. two groups have previously analyzed crispr-associated genes within e. faecalis and e. faecium genomes. partial crispr-like loci were previously described in e e and u0317; however, these loci were within a gene and were considered non-functional. in addition, palmer et al. identified crispr-cas predicted proteins in the broad institute strains com12;,733; and,. similarly, we only found a crispr-cas locus in strain tx out of the strains not previously studied. in summary, out of the available genomes, only one of the ha-clade isolates contained crisp-loci, namely the hybrid strain, the three other strains containing crispr-loci of the ca-clade all lacked antibiotic resistance determinants. therefore, our data coincide with the previous observation that members of the recently emerged high-risk enterococcal lineages lack crispr-loci and the inverse relationship between the presence of a crispr-cas locus and acquired antibiotic resistance. metabolic pathway metabolic pathways of e. faecium might have contributed to the recently increased incidence of e. faecium colonization and infection. to help understand e. faecium metabolism, the kegg pathway and kaas databases were used. both databases predicted more than pathways using tx genomic information. e. faecium exhibits major genomic differences in the genes involved in energy metabolism compared to that of other facultative anaerobic bacteria. however, like other species in the lactobacillaceae order, genes for typical aerobic energy generation through the tca cycle and electron transport chain do not exist, i.e., genes encoding complex i, ii, iii, and iv. when we compared the metabolic pathways of tx to those of e. faecalis v using the kegg database, all metabolic pathways of e. faecalis were also predicted in tx indeed, more diverse metabolic activities were observed in tx. additional files 10: table s and additional files 11: table s show lists of enzymes that only exist in e. faecium tx or e. faecalis v when kegg enzymes from both strains were compared. many of these enzymes were also described by van schaik et al. who compared european strains to e. faecalis v they found cogs present in their e. faecium genomes lacking in v whereas we found predicted enzymes present in tx lacking in e. faecalis v according to kegg analysis. additionally, they found cogs specific for e. faecalis v compared to the european strains, whereas we found only enzymes specific to v when compared to tx according to kegg analysis. plasmids alignment of orfs from the three plasmids of tx to the orfs from the other e. faecium genomes by blastp showed that all strains shared some orfs that are similar to the orfs of the three e. faecium tx plasmids, but none of them have more than 90% of the orfs from any of the plasmids. it is likely that some strains may have similar but not identical plasmids as tx but identification of plasmids in other strains is difficult since those genomes are draft sequences. alignment of orfs of the three tx plasmids to complete e. faecium plasmid sequences available in ncbi using tblastn with 90% identity and 50% match length cutoffs showed that pdo is most similar to plasmid pm7m a  kb plasmid which shared orfs of the orfs from pdo and that pdo is somewhat similar to plasmids prum and ps with % and % match to pdo orfs respectively. tx plasmid pdo does not seem to be similar to any completely sequenced e. faecium plasmids but has similarity to the partially sequenced e. faecium large plasmid plg both pdo and plg1plasmids harbor the hyaluronidase gene, the hylefm gene was also found in ha strains,,, c tc and u discussion tx was the first e. faecium strain sequenced and has been used in various studies since. the tx genome is characterized by numerous hyper variant loci and a large number of is elements and transposons. ortholog analysis as well as core and pan-genome analysis of tx and the other sequenced strains revealed that e. faecium genomes are highly heterogeneous in gene content and possess a large number of dispensable genes. similar to the findings by van schaik et al., pan and core genome analysis predict the pan genome to be open. phylogenetic analysis using single-copy orthologs of the same length and gene content dissimilarity analysis in addition to recent studies looking at core genes, snps and 16s rrna, all indicate a large divergence between ca-clade isolates and ha-clade isolates. furthermore, our previous analysis and analyses within this study show that cc genogroup isolates cluster more closely together and further away from the ca-clade isolates than the other non-cc ha-clade isolates, indicating the cc genogroup is a more recently evolved genogroup. genomic island analysis by codon usage bias and composition variation showed that tx has gis, although tx also possesses a large number of hyper variant loci, suggesting that most of the genomic variable loci in tx were acquired through lateral gene transfer, possibly through mobile elements such as transposons. in general, strains in the ha clade harbored more transposons than the ca strains and certain is elements such as is these findings are consistent with a previous study using whole genome microarray. although is presence has been proposed as an indicator of hospital-associated strains such as those apart of the cc genogroup, is was not found in all ha-clade strains. of note, however, all ha-clade strains contained the pbp5-r allele which may indicate that this is a reliable marker for hospital-associated isolates. indeed, the pbp5-r allele is also found in animal and community isolates that are considered within the ha-clade, but not considered clinically associated. the exception,, is interesting in that it is the ha-clade isolate from the blood of a hospitalized patient with no resistance genes, possibly supporting the concept that the genomic content of a strain, not just antibiotic resistance, adds to the survival in the hospital environment. in the gene analysis by galloway-pena et al., it was found that of the genes of this strain studied grouped with the community clade, indicating it is a hybrid strain as also reported in a recent study. capsular and other cell envelope polysaccharides of several gram-positive bacteria are known to have important roles in virulence and protective immunity. although the majority of studies on enterococcal surface polysaccharides have focused on e. faecalis, similar molecules have also been identified in e. faecium and suggested as targets for opsonic antibodies and as potential vaccine candidates, and also implicated in resistance of tx to phagocytosis in normal human serum. two such gene clusters, cps and epa, have been identified in e. faecalis. although a 7-9-gene cps region was recently determined necessary for the production of an e. faecalis capsular polysaccharide and shown to contribute to pathogenesis and evasion of the host innate immune response, tx only contains two homologs of the genes in this locus. in contrast, of the e. faecalis epa polysaccharide genes have homologs in tx and the other e. faecium genomes, although their sequences vary between the two species. therefore, it is likely that e. faecalis and e. faecium produce compositionally related, but not identical, epa surface polysaccharides. the hyper variable nature of the two polysaccharide loci found in tx raises the possibility that they are involved in biosynthesis of antigenically diverse surface polysaccharides which could help protect e. faecium against host immune responses. similar to other gram-positive bacteria, various mscramm-like cell wall anchored proteins have been previously identified in e. faecium; these include the collagen adhesin acm and biofilm-associated ebp pili, shown to be important for endocarditis and uti in animal models, respectively, as well as two other collagen-binding mscramms, scm and fms. our comparison of previously described mscramm and pilus encoding genes of tx with those of e. faecium draft genomes found them to be common among these strains and the majority of them to be enriched among ha clade strains or have a sequence variant mostly/exclusively carried by ca clade strains. thus, these findings agree with previous hybridization results and with the presence of two distinct subpopulations of e. faecium. furthermore, one of these genes, acm, was previously found to be expressed more often by clinical versus non-clinical isolates, whereas a pseudogene was often found in isolates from the community. taken together, these data indicate a clear difference in the mscramm and pilus gene profiles of the ha and ca clades, suggesting that these genes may have favored the emergence of ha-clade e. faecium in nosocomial infections. when we combined our finding with previously published results, four of the e. faecium genomes contain the crispr-cas locus. three of these strains are within the ca clade and lack all antibiotic resistances analyzed in this study. one of the strains,, is a unique strain in which its genome is a hybrid of ca and ha genes. however, it does have antibiotic resistance associated genes, showing there is not always an inverse relation between the number of antibiotic resistance determinants and the presence of crispr loci. more strains containing crispr-loci will need to be studied in order to determine if, is just an exception to the rule, or if the highly recombinant nature of e. faecium makes it different from e. faecalis with respect to the presence of crispr-loci in relation to antibiotic resistance determinants. overall, there seem to be some patterns that point to specific evolutionary events throughout e. faecium’s history as a species. first and foremost, there is a large ancestral split between the ca- and ha-clade strains which are separated by at least a 3–4% difference in their core genome. the ca-clade isolates, except one, do not have either polysaccharide synthesis locus or downstream of the epa region, antibiotic resistance genes, certain genomic islands, or is elements. after the ha-clade diverged from ca-clade there was further evolution within the ha clade and some ha-clade strains studied here may represent phylogenetic transitional lineages. like the ca-clade strains, these transitional lineages are characterized by a lack of is and have neither locus nor in the epa extension. although the data are limited, one scenario that could explain these observations is if locus replaced locus in a ha-clade ancestral strain, after the split from the ca clade, which later acquired is and then, subsequently, locus or replaced locus in the epa extension region. even if this is not the case, it seems clear that only strains further along in the phylogenetic trees, indicating a division within the ha-clade, acquired is and the polysaccharide biosynthesis loci and the exception is e a strain previously shown to have of genes from the ha-clade, which could have gained locus via recombination. also of note, three of the four strains that have locus downstream of the epa locus lack the ebp genes, possibly suggesting there may have been some kind of gain and loss through homologous recombination. figure  shows the projected scenarios for the evolution of the two clades of e. faecium as can be envisioned using our data as well as other previous publications. the hypothesis is that there was a primordial type of e. faecium which split many millinea ago and evolved into two early community groups which had homologous genes e.g. the pbp5-s or pbp5-r alleles, the latter representing community sources of are. these lineages could recombine with each other resulting in hybrid strains. the divergence between the two community groups eventually reached a core genomic difference of approximately 3–4%, creating a ha clade, which includes both ampicillin- resistant, community-based isolates, such as those from some canine and feline origins, as well as most of the clinical-, hospital- and outbreak- associated isolates and a ca clade, which consists mostly of community derived isolates. most likely, community and hospital are isolates split from the same ancestor, as represented by scenario two. however, it is also possible that are clones evolved from the animal reservoir, or that animal are isolates represent evolutionary descendants of hospital are transferred from humans to their pets.  in conclusion, the completion of the tx genome has provided insight into the intricate genomic features of e. faecium, and will surely serve as an important reference for those studying e. faecium genomics in the future. by studying tx an endocarditis isolate belonging to cc and comparing the tx genome to the other draft genomes, we have been able to confirm the high genomic plasticity of this organism. the ha-clade isolates contain a number of unique is elements, transposons, phages, plasmids, genomic islands, and inherent and acquired antibiotic resistance determinants, most likely contributing to the emergence of this organism in the hospital environment that has occurred in the last 30 years. 
in this study, we report the complete genome sequence of the e. faecium strain tx also known as do, which belongs to multilocus sequence type and was the first e. faecium strain ever sequenced. although the underlying mechanism for the gradual replacement of e. faecalis by e. faecium in the hospital environment is not yet understood, many studies using genotyping and phylogenetic analysis have shown the emergence of a globally dispersed polyclonal subcluster of e. faecium strains in clinical environments. orfs in tx are ha-clade specific and antibiotic resistance genes are enriched in ha-clade strains. which of these differences are important for the success of specific e. faecium lineages in the hospital environment remain to be determined. enterococci are among the leading causes of hospital-acquired infections in the united states and europe, with enterococcus faecalis and enterococcus faecium being the two most common species isolated from enterococcal infections. in the last decade, the proportion of enterococcal infections caused by e. faecium has steadily increased compared to other enterococcus species. mobile elements such as is and transposons were also found almost exclusively in ha strains, as previously reported. systematic study of the molecular epidemiology and pathogenesis of e. faecium has been hindered by the lack of closed, complete e. faecium genomes that can be used as references. whole genome comparison of the tx genome with e. faecium draft genomes confirmed that most clinical, outbreak, and hospital-associated strains, in addition to strains of non-hospital origin, group in the same clade and are evolutionally considerably more closely related to each other by phylogenetic and gene content similarity analyses than to isolates in the community-associated clade with approximately a 3–4% average nucleotide sequence difference between the two clades at the core genome level. our findings along with other studies show that ha clonal lineages harbor specific genetic elements as well as sequence differences in the core genome which may confer selection advantages over the more heterogeneous ca e. faecium isolates.
 enterococci are among the leading causes of hospital-acquired infections in the united states and europe, with enterococcus faecalis and enterococcus faecium being the two most common species isolated from enterococcal infections. in the last decade, the proportion of enterococcal infections caused by e. faecium has steadily increased compared to other enterococcus species. although the underlying mechanism for the gradual replacement of e. faecalis by e. faecium in the hospital environment is not yet understood, many studies using genotyping and phylogenetic analysis have shown the emergence of a globally dispersed polyclonal subcluster of e. faecium strains in clinical environments. systematic study of the molecular epidemiology and pathogenesis of e. faecium has been hindered by the lack of closed, complete e. faecium genomes that can be used as references.  in this study, we report the complete genome sequence of the e. faecium strain tx also known as do, which belongs to multilocus sequence type and was the first e. faecium strain ever sequenced. whole genome comparison of the tx genome with e. faecium draft genomes confirmed that most clinical, outbreak, and hospital-associated strains, in addition to strains of non-hospital origin, group in the same clade and are evolutionally considerably more closely related to each other by phylogenetic and gene content similarity analyses than to isolates in the community-associated clade with approximately a 3–4% average nucleotide sequence difference between the two clades at the core genome level. our study also revealed that many genomic loci in the tx genome are unique to the ha clade. orfs in tx are ha-clade specific and antibiotic resistance genes are enriched in ha-clade strains. mobile elements such as is and transposons were also found almost exclusively in ha strains, as previously reported.  our findings along with other studies show that ha clonal lineages harbor specific genetic elements as well as sequence differences in the core genome which may confer selection advantages over the more heterogeneous ca e. faecium isolates. which of these differences are important for the success of specific e. faecium lineages in the hospital environment remain to be determined. 