Bacterial strains and plasmids

Table 1 and Figure 1 show the strains and plasmids used in this work and depict the genetic organization of the hyl _Efm -region in E. faecium strains and mutants.

Strains/Plasmids	Relevant Characteristics	Reference
Strains
E. faecium
TX16 (DO)	Sequenced endocarditis clinical isolate, Emr, Smr. ST-16a http://www.hgsc.bcm.tmc.edu	[35]
TX1330RF	Fsr and Rfr derivative of TX1330, a faecal colonizing strain from a healthy human volunteer	[11]
TX1330RF (pHylEfmTX16)	Derivative of TX1330RF to which the hyl Efm -containing plasmid (pHylEfmTX16) was transferred by conjugation from TX16 (DO) (~250 kb)	[11]
TX1330RF (pHylEfmTX16Δ7,534)	Mutant with deletion of part or all of 6 genes of the hyl Efm region of TX1330RF(pHylEfmTX16)	This work
TX1330RF (pHylEfmTX16Δ4genes)	Non-polar deletion of 4 genes of the hyl Efm region of TX1330RF(pHylEfmTX16)	This work
TX1330RF (pHylEfmTX16Δ hyl )	Non-polar deletion mutant of hyl Efm of TX1330RF(pHylEfmTX16)	This work
TX1330RF (pHylEfmTX16Δ hyl-down )	Non-polar deletion of hyl Efm plus its downstream gene of TX1330RF(pHylEfmTX16)	This work
TX1330RF (pHylEfmTX16Δ down )	Non-polar deletion of the gene downstream of hyl Efm of TX1330RF(pHylEfmTX16)	This work
E. faecalis
CK111	OG1Sp upp4::P23 repA4	[25]
Plasmids
pHylEfmTX16	Conjugative and transferable megaplasmid (ca. 250 kb) of TX16 (DO) containing hyl Efm	[11]
pCJK47	Conjugative donor plasmid for markerless mutagenesis; oriTpCF10 and pheS* pORI280 derivative; confers Emr	[25]
pHOU1	Derivative of pCJK47 in which the erm(C) gene was replaced by aph-2'-ID; confers Gmr	This work
pHOU2	Derivative of pCJK47 in which the erm(C) gene was replaced by aph-2'-ID and cat was incorporated in the cloning site for allelic replacements; confers Gmr.	This work
pTEX5501ts	E. coli-enterococcal shuttle plasmid for mutagenesis using a temperature-sensitive replicon	[27]
pAT392	oriRpAMβ1, oriRpUC oriTRK2 spc lacZα P2 aac(6')-aph(2")	[30]
pAT392::hyl Efm	Derivative of pAT392 containing hyl Efm (cloned with SacI and SmaI) under the control of the P2 promoter	This work
pAT392::hyl Efm- down	Derivative of pAT392 containing both the hyl Efm plus downstream genes (cloned with SacI and SmaI) under the control of the P2 promoter	This work

Em^r, erythromycin resistance; Fs^r, fusidic acid resistance; Gm^r, gentamicin resistance, Rf^r, rifampin resistance; Sm^r, high-level resistance to streptomycin. ^aST refers to sequence type after multi-locus sequence typing. ST16 is part of CC17

Construction of a deletion mutant of the hyl _Efm -region using the pheS* counter-selection system in TX16(pHyl_EfmTX16) and its transfer to TX1330RF

The pheS* system (previously used in Enterococcus faecalis) [25] is based on the acquired sensitivity of bacteria to p-chloro-phenylalanine (p-Cl-Phe) if they carry a pheS* allele encoding a phenylalanine tRNA synthetase with altered substrate specificity [25, 26]. In order to apply this approach to E. faecium strains, which are commonly macrolide resistant, we constructed a derivative of the pheS* vector pCJK47 by replacing its erm(C) gene with aph2"-ID, which confers resistance to gentamicin. The full aph-2"-ID gene (including promoter and terminator regions) was amplified by PCR using plasmid pTEX5501ts [27] as the template with primers A and B (Table 2). The amplified fragment (1,089 bp) was digested with NsiI and BglII and ligated with pCJK47 digested with the same enzymes resulting in pHOU1 (Figure 2A). Subsequently, pHOU1 was digested with BamHI and PstI and ligated with a 992 bp fragment released from pTEX5501ts after digestion with the same enzymes and containing the chloramphenicol acetyl-transferase gene (cat), obtaining a 7,906 bp vector designated pHOU2 (Figure 2B).

Primer	Sequence (5'-3')	Relevant Characteristics
A	gaagatctgagataggttatgcaagat	Forward, BglII site (underlined), used amplification of aph-2"-ID
B	ccaatgcatgattccggattctaaaaaagg	Reverse, NsiI site (underlined), used amplification of aph-2"-ID
C	cgggatccgtttaaaaccagctggaaaag	Forward, BamHI site (underlined), located 1,251 nucleotides upstream of the start codon of the gene encoding a putative glycosyl hydrolase family 20 (Figure 1.)
D	ccgctcgagcaattcaacattgcaaagac	Reverse, XhoI site (underlined), located 294 nucleotides upstream of the start codon of the gene encoding a putative glycosyl hydrolase family 20 (Figure 1.)
E	cgagggcccgtgaagtattgccagatgt	Forward, ApaI site (underlined); located 592 nucleotides downstream of the down gene (hypothetical, Figure 1.)
F	ccggaattcaaaagcagaattggaaatca	Reverse, EcoRI site, 1,571 nucleotides downstream of the down gene (hypothetical, Figure 1.)
G	gcgagctcgattactttcaaaggaga	Forward, SacI site (underlined), ribosomal binding site of hyl Efm (italics) (Figure 1.)
H	tcccccggg ctaacttttgataatttgctc	Reverse, SmaI site, (underlined) and stop codon of hyl Efm (Figure 1.)
I	tcccccggg ttagcgattgatcgagc	Reverse, SmaI site (underlined), stop codon of down (Figure 1.)
J	cgggatcccaatcaagaagtagcggatt	Forward, BamH site (underlined) 438 nucleotides upstream of the stop codon of carbohydrate ABC transporter gene (Figure 1.)
K	gcggccgctcgagggcccttagtgcgattgtatctgac	Reverse, stop codon of the gene that encodes to transmembrane protein (Figure 1.)
L	gggcccctcgaggcggccgcaaaattaaataaaaaatgg	Forward, ApaI, XhoI, NotI site, stop codon down (Figure 1.)
M	catgcatgaatcaggaactgaaactgc	Reverse, NsiI site, 1,091 nucleotides upstream of stop codon of GMP synthase (opposite orientation) (Figure 1.)
N	ccggaattccagtaaaaggcacagagc	Forward, EcoRI site (underlined), located 2,138 nucleotides down-stream of glycosyl hidrolase family 45-2 start codon (Figure 1.)
O	tcatctattttctcctttgaaagtaatcactatattcc	Reverse, stop codon of glycosyl hydrolase family 45-2 (Figure 1.)
P	tcaaaggagaaaatagatgaatatcttaaaaaataaaaagc	Forward, located 40 nucleotides upstream of down gene start codon (Figure 1.)
Q	ataagaatgcggccgcttagcgattgatcgagcg	Reverse, NotI site (underlined), stop codon of down (Figure 1.)
R	ataagaatgcggccgccagtaaaaggcacagagc	Forward, NotI site (underlined), located 2,138 nucleotides down-stream of glycosyl hydrolase family 45-2 start codon (Figure 1.)
S	tcatctattttctcctttgaaagtaatcactatattcc	Reverse, stop codon of glycosyl hydrolase family 45-2 (Figure 1.)
T	tcaaaggagaaaatagatgacaaaattaaataaaaaatgg	Forward, 1,973 nucleotides upstream of stop codon of GMP synthase (Figure 1.)
U	cggaattcgaatttgtatatgtcttcg	Reverse, EcoRI site (underlined), 994 nucleotides upstream of start codon of GMP synthase (opposite direction) (Figure 1.)
V	aaggaaaaaagcggccgccagaatatgataatcgtcatgg	Forward, NotI site (underlined), 902 nucleotides downstream of hyl Efm start codon (Figure 1.)
W	tttgttctcctttttcttgctttttattttttaag	Reverse, stop codon of of hyl Efm (Figure 1.)
X	gcaagaaaaaggagaacaaacaaaattaaataaaaaatgg	Forward, 1,973 nucleotides upstream of stop codon of GMP synthase (opposite direction) (Figure 1.)
Y	ccggaattcgaatcaggaactgaaactgccc	Reverse, EcoRI site (underlined), 1,094 nucleotides upstream of stop codon of GMP synthase (opposite direction) (Figure 1.)
A1	cgcgtcgtattaaaaatcat	Forward, 143 nucleotides upstream of stop codon of GH20 (Figure 3.)
A2	gatcgataaactggctcgt	Reverse, 139 nucleotides upstream of start codon of GH42 (Figure 3.)
B1	acgcgtcgacagcagctggatatgctga	Forward, SalI site (underlined), 2,316 nucleotides downstream of start codon of GH42 (Figure 3.)
B2	ggaagatctccggtttccagacttctt	Reverse, BglII site (underlined), 159 nucleotides downstream of start codon of hyl Efm (Figure 3.)
C1	gttagaagaagtctggaaaccg	Forward, 138 nucleotides downstream of start codon of hyl Efm (Figure 3.)
C2	tgctaagatattcctctactcg	Reverse, 798 nucleotides upstream of stop codon of hyl Efm (Figure 3.)
D1	acatgcatgcagaattggagccttggtt	Forward, SphI site (underlined), 169 nucleotides upstream of stop codon of hyl Efm (Figure 3.)
D2	cggaattctgcttccgcataagaaa	Reverse, EcoRI site (underlined), 319 nucleotides upstream of stop codon of down gene (Figure 3.)
E1	gcaaggcttcttagaga	Forward, ddl E. faecium [32, 33]
E2	catcgtgtaagctaacttc	Reverse, ddl E. faecium [32, 33]

In order to create a deletion mutant of the hyl _Efm -region (which contains genes predicted to be involved in carbohydrate metabolism and transport; Figure 1), fragments upstream (977 bp) and downstream (999 bp) of this region were amplified by PCR (with primers C-D and E-F, respectively; Table 2) and cloned upstream and downstream of the cat gene in pHOU2, respectively, using BamHI and XhoI for the upstream fragment and ApaI and EcoRI for the downstream fragment; the correct insert was confirmed by sequencing in both directions. This recombinant plasmid was introduced into E. faecalis CK111 by electroporation as described previously [25, 28] and blue colonies were recovered on brain heart infusion (BHI) agar plates containing gentamicin (125 μg/ml) and X-Gal (200 μg/ml). Subsequently, the pHOU2 derivatives were introduced into strain TX16 by filter mating [29] with E. faecalis CK111 as the donor. Single cross-over integrants were selected on gentamicin (170 μg/ml) and erythromycin (200 mg/ml) and purified colonies were then resuspended in 50 μl of normal saline and plated on MM9YEG media (salts and yeast extract) supplemented with 7 mM of p-Cl-Phe [25] and incubated for 48 h at 37°C. To confirm that colonies which grew on MM9YEG media supplemented with p-Cl-Phe were excisants, the corresponding colonies were grown simultaneously on BHI agar in the presence and absence of gentamicin. Colonies that were susceptible to gentamicin were further screened by PCR, pulsed field gel electrophoresis (PFGE) and hybridizations with hyl _Efm and cat probes as described before [11]. The mutated region was also sequenced in order to confirm deletion of the corresponding genes. Subsequently, the mutated hyl _Efm -containing plasmid (pHyl_{EfmTX16Δ7,534}) was transferred from E. faecium TX16 to TX1330RF (a fusidic and rifampin resistant derivative of the commensal strain TX1330, Table 1) by filter mating as described previously [11] to obtain the strain TX1330RF(pHyl_{EfmTX16Δ7,534}). Acquisition of the mutated plasmid by TX1330RF was also confirmed by PFGE, PCR, hybridizations and sequencing. S1 nuclease digestion and PFGE was performed with the mutant to confirm that no other plasmid had transferred during the conjugation event as previously described [11].

Complementation of the hyl _Efm -region mutant TX1330RF(pHyl_{EfmTX16Δ7,534})

The hyl _Efm gene was PCR amplified with primers G and H (including the ribosomal binding site and the stop codon of hyl _Efm ) (Table 2) using total DNA from TX16 as template, and the DNA fragment (1,685 bp) cloned into the shuttle plasmid pAT392 [30] under the control of the P2 promoter (which allows constitutive expression of the cloned genes) and upstream of the aac(6')-aph(2") gene (which is co-transcribed from the same promoter) using SacI and SmaI sites (plasmid pAT392::hyl _Efm ). In order to evaluate if the deletion of hyl _Efm had an effect in the downstream gene (encoding a hypothetical protein of 331 amino acids of unknown function), the hyl _Efm and down genes (Figure 1) were also cloned together into pAT392 following a similar strategy and using primers G and I (pAT392::hyl _Efm -down). Recombinant pAT392-derivatives were purified from E. coli grown on Luria-Bertani agar containing gentamicin (25 μg/ml) and all their DNA inserts sequenced. Subsequently, they were introduced into E. faecium TX1330RF, and the TX1330RF(pHyl_{EfmTX16Δ7,534}) mutant by electroporation. Stability of the plasmid constructs was tested by isolating ca. 100 colonies from overnight cultures (using BHI broth) and from the spleens of dead animals (in different experiments) after intraperitoneal inoculation of the corresponding strain (see below) and plating them simultaneously on BHI and BHI-gentamicin (125 μg/ml).

Construction of additional mutants of the hyl _Efm -region in E. faecium TX1330RF(pHyl_EfmTX16)

To investigate the specific role of the hyl _Efm locus in E. faecium pathogenesis, complete in-frame deletions of four genes of the hyl _Efm -region, hyl _Efm alone, hyl _Efm plus its downstream gene and the gene downstream of hyl _Efm were generated using TX1330RF(pHyl_EfmTX16). Fragments upstream and downstream of each region were amplified by PCR with the corresponding primers (Figure 1 and Table 2). These fragments, with overlapping ends, were subsequently amplified by crossover PCR and cloned into pHOU1 using EcoRI and NotI (for hyl _Efm , hyl _Efm plus its downstream gene and the downstream gene of hyl _Efm mutants); and BamHI and PstI (for the four gene mutant). The inserts were sequenced in both directions to confirm that no mutations had been introduced during the cloning process. The recombinant plasmids were electroporated or transferred by conjugation (using E. faecalis CK111) into TX1330RF(pHyl_EfmTX16). Single crossover events and deletions of targeted regions (Figure 1) were obtained by plating in BHI with gentamicin and p-Cl-Phe containing medium, respectively, as previously described [25]. Confirmation of the deletion was performed by PCR, PFGE, hybridizations and DNA sequencing.

RT-PCR

RNA was extracted from bacterial cells (TX16, TX1330RF(pHyl_EfmTX16), TX1330RF and strains containing pAT392 derivatives) grown in BHI broth at 37°C with mild agitation (logarithmic phase of growth, A₆₀₀ 0.8) as described before [31], and using the RNA isolation kit RNAwiz (Ambion, Austin, TX). RNA was treated twice with DNase (DNase-Free solution, Ambion) and synthesis of cDNA was performed using the commercial kit SuperScript One-Step reverse transcription-PCR (RT-PCR) with Platinum Taq (Invitrogen), according to the manufacturer's instructions. The mixture contained 0.2 μM of each primer, designed to detect overlapping transcripts of the four putative metabolic genes (Figure 3) and an internal transcript of hyl _Efm (Table 2). A primer pair directed to detect a 550-bp transcript of the housekeeping gene ddl _{E. faecium} was used as an internal control for RT-PCR experiments [32, 33].

Mouse peritonitis model

Female (4 to 6 week old), outbred ICR mice (Harlan Sprague Dawley, Houston) were used as previously described [34]. Groups of 10 mice per inoculum (ranging from 2.3 × 10⁸ to 3.1 × 10⁹ CFU/ml) were included in each experiment. Inocula for each peritonitis experiment were prepared by growing bacteria initially on BHI agar plates. Subsequently, one colony was grown in BHI broth for 24 h at 37°C and the cells were concentrated in saline (0.9%) to an A₆₀₀ of ca. 1.2. Strains containing pAT392 and derivatives were handled similarly before the intraperitoneal inoculation, except that the BHI agar and broth contained gentamicin (125 μg/ml). Comparison of the survival curves at similar inocula was performed using a log-rank test with Prism for Windows^®. A P < 0.05 was considered significant. All experiments were approved by the Animal Welfare committee, University of Texas Health Science Center at Houston.

Deletion of 6 genes in the E. faecium hyl _Efm -region altered in vitro growth and attenuated virulence of TX1330RF(pHyl_EfmTX16) but not TX16(pHyl_EfmTX16) in murine peritonitis

Since acquisition of the transferable pHyl_EfmTX16 by TX1330RF conferred increased virulence in experimental peritonitis [11], we explored the possibility that the hyl _Efm region was an important mediator of this effect. Using RT-PCR assays, we were able to detect in vitro expression of hyl _Efm during the exponential phase of growth in both TX16 and TX1330RF (pHyl_EfmTX16) (Figure 3). RT-PCR with primers located at the 3' and 5' ends of contiguous genes yielded products of the expected size in each case, suggesting that these genes are likely to be co-transcribed (Figure 3). Then, we adapted the pheS* counter-selection system [25] developed for E. faecalis to obtain several deletions of the hyl _Efm -region. The hyl _Efm gene in E. faecium TX16 (http://www.ncbi.nlm.nih.gov/genomeprj/30627 Genbank accession number ACIY00000000) is located in a cluster of genes whose putative function appears to involve the transport and breakdown of carbohydrates (Figure 1) [13]. As an initial step to test the mutagenesis system, a relatively large deletion (7,534 bp) from pHyl_EfmTX16 was obtained. The deletion involved three genes predicted to encode glycosyl hydrolases (including hyl _Efm ) and a gene downstream of hyl _Efm whose function is unknown (Figure 1). Part (226 nucleotides) of a gene encoding a hypothetical transmembrane protein and located upstream of the putative family 20 glycosyl hydrolase gene and part (202 nucleotides) of a gene located 1,332 nt downstream of hyl _Efm encoding a putative GMP-synthase and likely transcribed in the opposite direction from the hyl _Efm cluster (Figure 1) were also deleted. As it is shown in Figure 4A, the deletion of 7,534 bp in the hyl _Efm -region did not affect the virulence of TX16 (DO) in murine peritonitis.

Next, we considered the possibility that an in vivo effect might be more clearly dissected if studies were performed in the background of a non-clinical strain. We hypothesized that an in vivo effect of a virulence determinant might more likely be seen in strains which are less successful clinically; that is, that a commensal strain such as TX1330RF [11] is likely to have decreased fitness or ability to produce disease compared to TX16 [35] and, thus, acquisition plus subsequent loss of a virulence determinant that alters such fitness would be easier to identify [11]. Thus, the mutated plasmid from strain TX16(pHyl_{EfmTX16Δ7,534}) was transferred to TX1330RF by conjugation and the in vivo effect of acquiring the intact plasmid [11] vs the plasmid carrying the deletion was evaluated. The two strains [TX1330RF(pHyl_EfmTX16) and TX1330RF(pHyl_{EfmTX16Δ7,534})] appeared to differ only in the size of the hyl _Efm plasmid by PFGE and S1 nuclease assays [11] (not shown). Figure 4B shows that deletion of 7,534 bp in the hyl _Efm region of TX1330RF(pHyl_EfmTX16) caused an in vitro growth defect. The alteration of growth was also seen in a second transconjugant from the same mating experiment between TX16(pHyl_{EfmTX16Δ7,534}) and TX1330RF (TC-II in Figure 4B). The mutant strain TX1330RF(pHyl_{EfmTX16Δ7,534}) was attenuated in the mouse model of peritonitis (even when an increased intraperitoneal inoculum for the mutant were used) (Figure 4C and 4D) (P < 0.05). Due to the alterations produced in the growth of TX1330RF(pHyl_{EfmTX16Δ7,534}), these results suggest that the attenuation in virulence may have also been due to factors other than those specifically related to virulence.

Complementation of the hyl _Efm -region mutant with hyl _Efm and a combination of hyl _Efm and the downstream gene did not restore the virulence of TX1330RF(pHyl_{EfmTX16Δ7,534})

In order to further evaluate if the attenuation observed in TX1330RF(pHyl_{EfmTX16Δ7,534}) (as described above) was mediated by a direct effect of hyl _Efm in the peritonitis model, we explored complementation of this mutant in trans with the full hyl _Efm gene and a combination of hyl _Efm and the downstream gene using the shuttle vector pAT392 [30]. The cloning strategy placed these genes upstream of the aac(6')-aph(2") gene (which confers resistance to gentamicin) resulting in all open reading frames under the control of the constitutive P2 promoter. Up to 80% loss was observed with all strains in the absence of gentamicin; however, in the presence of the antibiotic during inoculum preparation, the TX1330RF(pHyl_{EfmTX16Δ7,534})-derivatives containing the pAT392 constructs were stable both in vitro and in vivo (5% maximum percentage of plasmid loss). Introduction of hyl _Efm or a combination of hyl _Efm plus its downstream gene (cloned into pAT392) did not restore the virulence of the mutant strain TX1330RF(pHyl_{EfmTX16Δ7,534}), compared to pAT392 alone in the presence of gentamicin (Figure 5A and 5B). The results indicate that constitutive expression of hyl _Efm alone or in combination with its downstream gene (which was confirmed by RT-PCR, not shown) was not able to restore the phenotypic differences observed in the mutant strain TX1330RF(pHyl_{EfmTX16Δ7,534}), supporting the fact that hyl _Efm may not be directly responsible of the attenuation observed in the mutant.

Under our experimental conditions, we cannot completely rule out that the in vivo attenuation observed with pHyl_{EfmTX16Δ7,534} in the TX1330RF background may have been caused by the partial deletion of the hypothetical transmembrane protein or the putative GMP-synthase located upstream and downstream of the hyl _Efm -cluster, respectively. Indeed, a deletion of 76 amino acids in the C-terminus of the hypothetical membrane protein occurred in this plasmid, resulting in the deletion of three predicted transmembrane helices. Similarly, 68 amino acids in the C-terminus of the putative GMP-synthase were deleted; the removal of these amino acids is likely to disturb the dimerization domain of this protein [36] affecting its function in nucleotide metabolism. Moreover, a second TX1330RF(pHyl_{EfmTX16Δ7,534}) mutant also exhibited an almost identical growth defect (Figure 4B). Thus, it is tempting to speculate that changes in these two genes may have affected the "metabolic" fitness of the TX1330RF(pHyl_{EfmTX16Δ7,534}) strain. However, since no evident change in fitness or virulence was observed with the mutated plasmid in the TX16 background, another possibility is that an extraneous change elsewhere in the plasmid (or chromosome) occurred during the conjugation process that influenced the in vitro growth of the TX1330RF(pHyl_{EfmTX16Δ7,534}) mutant(s) and its virulence.

Additional deletions of genes in the hyl _Efm -region did not alter the virulence of TX1330RF(pHyl_EfmTX16) in the mouse peritonitis model

In order to dissect further the in vivo role of hyl _Efm and the adjacent genes, we produced several in-frame deletions of these genes (Figure 1) including: i) a four gene mutant of the hyl _Efm -region (including hyl _Efm ) [TX1330RF(pHyl_{EfmTX16Δ4genes})], ii) a deletion of hyl _Efm alone [TX1330RF (pHyl_{EfmTX16Δ hyl} )], iii) a deletion of hyl _Efm plus its downstream gene mutant [TX1330RF (pHyl_{EfmTX16Δ hyl-down} )] and, iv) a single deletion of the gene located downstream from hyl _Efm [TX1330RF (pHyl_{EfmTX16Δ down} )]. The mutagenesis strategy removed the open reading frame from the start codon of the first gene to the stop codon of the last gene (in case of multiple genes). In case of single gene deletion, the complete ORF (start to stop codon) was removed, leaving the surrounding DNA intact as in the wild type plasmid. None of the four mutants of the hyl _Efm -region showed a deleterious effect in the growth kinetics compared to TX1330RF (pHyl_EfmTX16) (harbouring an intact plasmid, Additional file 1). Moreover, we were unable to observe any attenuation of virulence in the mouse peritonitis model compared to the parental strain with the intact plasmid (Figure 6A-D), which further supports the fact that the four genes of the hyl _Efm region do not appear to be directly involved in increasing the pathogenic potential of pHyl_EfmTX16 in strain TX1330RF(pHyl_EfmTX16).

Megaplasmids (>145 kb, with or without hyl _Efm ) have been recently found to be widespread among clinical isolates of E. faecium worldwide [12, 13, 15]. The proportion of these plasmids carrying hyl _Efm appears to vary according to geographical location (ca. 11 to 36%) [12, 13]. Our findings indicate that the four genes of the hyl _Efm -cluster studied here, including hyl _Efm are not the main mediators of the virulence effect conferred by the plasmid carrying them in experimental peritonitis. Since the pHyl_Efm plasmids are large, it is presumed that other genes (i.e., upstream or downstream of the glycoside hydrolase-encoding genes) are more relevant in mediating this effect. Additionally, we cannot exclude that the hyl _Efm cluster studied in this work may play a role in other infections such as endocarditis or urinary tract infections (a subject of our ongoing studies). As a final remark, the adaptation of the pheS* counter-selection system for targeted mutagenesis in plasmid and chromosomal genes of E. faecium will facilitate the understanding of the role of other specific plasmid genes in the pathogenesis of E. faecium infections in the near future.