JOURNAL OF BACTERIOLOGY, Aug. 2005, p. 5709–5718
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 187, No. 16
Molecular Characterization of a Widespread, Pathogenic, and
Antibiotic Resistance-Receptive Enterococcus faecalis
Lineage and Dissemination of Its Putative
Sreedhar R. Nallapareddy,1,2Huang Wenxiang,1,2† George M. Weinstock,3,4
and Barbara E. Murray1,2,3*
Division of Infectious Diseases, Department of Internal Medicine,1Center for the Study of Emerging and
Re-emerging Pathogens,2and Department of Microbiology and Molecular Genetics,3University of
Texas Medical School, and Human Genome Sequencing Center,
Baylor College of Medicine,4Houston, Texas 77030
Received 21 March 2005/Accepted 25 May 2005
Enterococcus faecalis, a common cause of endocarditis and known for its capacity to transfer antibiotic
resistance to other pathogens, has recently emerged as an important, multidrug-resistant nosocomial patho-
gen. However, knowledge of its lineages and the potential of particular clones of this species to disseminate and
cause disease is limited. Using a nine-gene multilocus sequence typing (MLST) scheme, we identified an
evolving and widespread clonal complex of E. faecalis that has caused outbreaks and life-threatening infections.
Moreover, this unusual clonal complex was found to contain isolates of unexpected relatedness, including the
first known U.S. vancomycin-resistant enterococcus (E. faecalis strain V583), the first known penicillinase-
producing (Bla?) E. faecalis isolate, and the previously described widespread clone of penicillinase producers,
a trait found in <0.1% of E. faecalis isolates. All members of this clonal cluster (designated as BVE for Bla?
Vanrendocarditis) were found to contain a previously described putative pathogenicity island (PAI). Further
analysis of this PAI demonstrated its dissemination worldwide, albeit with considerable variability, confirmed
its association with clinical isolates, and found a common insertion site in different clonal lineages. PAI
deletions, MLST, and the uncommon resistances were used to predict the evolution of the BVE clonal cluster.
The finding of a virulent and highly successful clonal complex of E. faecalis with different members resistant
to the primary therapies of choice, ampicillin and vancomycin, has important implications for the evolution of
virulence and successful lineages and for public health monitoring and control.
Enterococcus faecalis, a natural inhabitant of the gastroin-
testinal tract and a known cause of infective endocarditis since
ca. 1900 (14), has more recently emerged as a significant nos-
ocomial pathogen (12). Interest in enterococci derives in part
because of their prominence in multidrug-resistant nosocomial
infections (15), which are difficult to control or treat, their
propensity for incorporation of mobile elements (28) and their
ability to transfer these resistance phenotypes to other patho-
gens, including the apparent transfer of vancomycin resistance
from E. faecalis to methicillin-resistant Staphylococcus aureus
in humans (39). The present understanding about the clonal
relationships of E. faecalis isolates is limited to sporadic out-
break studies, and further knowledge about its population
structure is important for understanding what makes this or-
Molecular typing has shown that discrete lineages of patho-
genic bacteria can arise periodically and then spread locally or
globally in the presence of strong selective pressure (26). Thus,
the identification of E. faecalis clones that are successful in
achieving prolonged, widespread outbreaks and the unraveling
of their genetic background may shed light on the question of
how this opportunist adapts to clinical settings and behaves as
a pathogen, causing a range of infections such as intraabdomi-
nal, genitourinary, endovascular, or meningeal infections,
among others. Among typing methods for examining related-
ness of bacterial genetic backgrounds, multilocus sequence
typing (MLST), which is objective and less prone to human
error, has gained recognition as one of the best approaches and
has been used to identify pathogenic lineages of several spe-
cies, including Neisseria meningitidis, Streptococcus pneu-
moniae, S. aureus, and Enterococcus faecium (4, 7, 10, 26),
among others. Recently, we derived a four-gene MLST system
using one housekeeping gene (pyrC) and three antigen-encod-
ing genes (ace, efaA, and salA), chosen for their likely greater
variation, which successfully differentiated E. faecalis at the
subspecies level (23). When we subsequently used this MLST
scheme to examine additional selected isolates from our 30-
year collection, the results suggested an unexpected relation-
ship among clinically important isolates disseminated in sev-
eral states of the United States. We decided to further
investigate these isolates using additional housekeeping genes
plus the antigen-encoding genes; this combined use of different
types of genes has the potential advantage of revealing both
* Corresponding author. Mailing address: Div. Infectious Diseases,
Dept. Internal Medicine, Center for the Study of Emerging and Re-
emerging Pathogens, University of Texas Medical School at Houston,
6431 Fannin Street, MSB 2.112, Houston, TX 77030. Phone: (713)
500-6745. Fax: (713) 500-6766. E-mail: email@example.com.
† Present address: Department of Infectious Diseases, Chongqing
University of Medical Sciences, Chongqing, China 400016.
the long-term evolutionary history of the chromosome and a
short-term differentiation resulting from the more variable an-
tigen-encoding genes. After identifying a circulating E. faecalis
lineage that had acquired resistances to the primary and sec-
ondary drugs of choice, ampicillin and vancomycin, we further
explored the virulence gene profiles and predicted the evolu-
tion of this clonal complex based on acquired resistance genes
and variations observed in the previously reported pathogenic-
ity island (PAI) (34).
(A part of this work was presented at the 43rd Interscience
Conference on Antimicrobial Agents and Chemotherapy,
2003, Chicago, Ill.)
MATERIALS AND METHODS
Bacterial isolates. Twenty-one E. faecalis isolates that were recovered from a
broad geographic region, including previously defined ?-lactamase-producing
(Bla?) isolates (5, 11, 13, 17, 20, 23, 27, 33, 38), were chosen for this study based
on preliminary results suggesting an unexpected relatedness of some and because
of the general interest to the field of others (e.g., V583, MMH594, OG1RF, and
JH2-2) (5, 8, 11, 21, 23, 28, 32, 34, 38). Relevant background and characteristics
are detailed in Table 1. ?-Lactamase production was reconfirmed using nitroce-
fin disks. To assess the widespread nature of PAI (34), a total of 454 E. faecalis
strains isolated over 30 years from diverse locations (United States, Thailand,
China, Argentina, Chile, Spain, Canada, Belgium, United Kingdom, France, and
Lebanon), including nosocomial clinical isolates, nosocomial- and community-
derived fecal isolates, and animal isolates, were included. The “other clinical”
group includes isolates from blood, bile, bone, catheters, cervix, cerebrospinal
fluid, placenta, peritoneal fluid, sputum, and several types of wounds, among
others. In this extensive collection, most of the E. faecalis strains were typed
previously by pulsed-field gel electrophoresis (PFGE), and isolates with identical
patterns were excluded for the analysis of PAI presence in distinct clones.
Genomic DNA isolation, PCR, and DNA sequencing. E. faecalis isolates freshly
streaked from freezer vials onto brain heart infusion agar (Difco Laboratories,
Detroit, Mich.) were cultured in brain heart infusion broth. Genomic DNA was
extracted by the hexadecyltrimethyl ammonium bromide method as described
previously (41). The ef numbers used in this study are from the V583 genome
annotation (28). Internal fragments of three antigen-encoding genes (ace, en-
coding a collagen and laminin adhesin; efaA, encoding an endocarditis antigen;
and salA, encoding a cell wall-associated antigen) and six housekeeping genes
TABLE 1. Enterococcus faecalis strains by source of isolation, PFGE type, and nine-gene MLST
Clinical source; origin; yr of
Nine-gene (allelic profile for ace, efaA,
salA, pyrC, gki, gdh, aroE, xpt, and
TX0052 Endocarditis; Springfield, Mo.;
5b ST-15 (3, 3, 8, 6, 7, 3, 6, 1, 5) 11
Blood; St. Louis, Mo.; 1987
Blood; Madison, Wis.; 1985
ST-14 (3, 3, 8, 2, 7, 3, 6, 1, 5)
ST-14 (3, 3, 8, 2, 7, 3, 6, 1, 5)
Urine; Richmond, Va.; 1990
Sputum; Richmond, Va.; 1991
Pittsburgh, Pa.; 1987
Jacksonville, Fla.; 1989
Durham, N.C.; 1991
ST-6 (3, 5, 8, 2, 7, 3, 6, 1, 5)
ST-6 (3, 5, 8, 2, 7, 3, 6, 1, 5)
ST-6 (3, 5, 8, 2, 7, 3, 6, 1, 5)
ST-6 (3, 5, 8, 2, 7, 3, 6, 1, 5)
ST-6 (3, 5, 8, 2, 7, 3, 6, 1, 5)
11, 23, 33, 38
23, 33, 38
5, 11, 20, 38
5, 20, 38
PENN (TX0669, PA)d
Wilmington, Del.; 1986
Blood; Philadelphia, Pa.; 1983
Urine; Houston, Tex.; 1981
ST-7 (3, 5, 9, 2, 7, 3, 6, 1, 5)
ST-7 (3, 5, 9, 2, 7, 3, 6, 1, 5)
ST-7 (3, 5, 9, 2, 7, 3, 6, 1, 5)
5, 11, 20, 23, 38
5, 20, 23, 38
5, 11, 20, 23, 38
Blood; Houston, Tex.; 1996HH6ST-2 ST-2 (3, 3, 2, 2, 2, 6, 6, 1, 5)1, 23
Urine; Houston, Tex.; 19941 ST-12ST-12 (8, 3, 2, 2, 2, 6, 6, 1, 5) 1, 11, 23
Blood; Argentina; 1989 19ST-9 ST-9 (7, 7, 3, 1, 4, 7, 1, 1, 4) 5, 11, 17, 20, 23, 38
Urine; West Haven, Conn.; 19868 ST-13 ST-13 (9, 7, 4, 1, 4, 7, 1, 1, 4) 5, 20, 23, 27, 38
OG1RF (TX4002) Laboratory strain; ?1978g
VIII ST-1ST-1 (1, 1, 1, 1, 1, 1, 1, 1, 1)5, 11, 23, 38
BE83 (TX0855)Urine; Thailand; 1980 B-1ST-4 ST-4 (5, 6, 1, 1, 5, 5, 4, 4, 1)5, 19, 23
Chicken product; Spain; 1998BR-1ST-5ST-5 (4, 9, 1, 2, 1, 3, 1, 3, 1) 23, 31
JH2-2 (TX4000) Laboratory strain; ?1974g
VIIST-8ST-8 (4, 2, 7, 2, 6, 4, 5, 5, 4) 5, 11, 23, 38
Blood; Beirut, Lebanon; 19897ST-10 ST-10 (6, 4, 4, 5, 4, 2, 3, 2, 2) 20, 23, 38
BE88 (TX0860)Catheter tip; Thailand; 1980 B-3ST-11 ST-11 (7, 8, 6, 3, 3, 7, 2, 1, 3)5, 19, 23, 38
aAs designated in previous studies.
bThe MLST and PFGE pattern names from the earlier publication(s) are used.
eIsolates E228 and E366 were from the same hospital.
fIsolates HH22, TX2621, and TX2486 were from the same hospital.
gThe exact years of isolation of these two commonly used laboratory strains are unknown.
5710 NALLAPAREDDY ET AL.J. BACTERIOL.
(pyrC, ef1718 coding for dihydroorotase; gki, ef2788 coding for glucokinase; gdh,
ef1004 coding for glucose-6-phosphate 1-dehydrogenase; aroE, ef1561 coding for
shikimate 5-dehydrogenase; xpt, ef2365 coding for xanthine phosphoribosyltrans-
ferase; and yqiL, ef1364 coding for acetyl coenzyme A acetyltransferase) were
amplified using the optimized buffer B (1? buffer: 60 mM Tris-HCl [pH 8.5], 15
mM ammonium sulfate, and 2 mM MgCl2) obtained from Invitrogen (Carlsbad,
Calif.). PCR was performed in volumes of 50 ?l, with an initial denaturation at
94°C for 2 min followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for
30 s to 1 min (depending on size of the amplicon) and a final extension of 72°C
for 7 min. The PCR primers used for amplification and sequencing of all nine
genes are listed in Table 2. PCR amplicons purified using the Wizard PCR DNA
Cleanup system (Promega Corporation, Madison, Wis.) were sequenced using an
Applied Biosystems Prism 377 automated DNA sequencer using the Taq dye-
deoxy terminator method (PE Applied Biosystems, Foster City, Calif.). Se-
quences were assembled using the SeqMan program of DNASTAR software
(Lasergene, Madison, Wis.).
Sequence analysis, allele and ST assignment, and data analysis. Sequence
alignments for the nine gene fragments were done by the Jotun Hein method (6)
using the MegAlign program of DNASTAR software. To identify nucleotide
variation, sequences from the different isolates were compared to the corre-
sponding sequences in the well-studied E. faecalis strain OG1RF (21). For each
locus, every sequence with ?1 nucleotide change was classified as a distinct allele
(no weight was given to the degree of sequence divergence between alleles) and
each isolate was defined by its allelic profile (a series of numbers corresponding
to the alleles at the nine loci). In keeping with other studies, isolates with the
same nine allelic profiles were assigned the same sequence type (ST), and
isolates that shared alleles at ?7 loci (single or double locus variants [SLVs or
DLVs]) were called a clone. For linked STs differing by SLVs, the term clonal
complex was used, with the implication that these are descendants of a common
ancestor. Clonality was assessed using BURST, a clustering algorithm designed
for use with MLST data sets of bacterial pathogens (9).
PFGE and hybridizations. PFGE was performed with some modifications of a
previously described method (19). Agarose plugs containing genomic DNA
were digested with SmaI (Invitrogen), and electrophoresis was carried out
using clamped homogeneous electric field (CHEF-DRII; Bio-Rad Laborato-
ries, Richmond, Calif.), with ramped pulse times beginning with 5 s and
ending with 45 s, at 200 V for 26 h. The PFGE patterns were interpreted using
the criteria suggested by Tenover et al. (37), with closely and possibly related
patterns being designated as belonging to a single clone. PFGE pattern names
that were presented in earlier publications are used here. Southern and
colony lysate hybridizations were performed under high-stringency conditions
(36) with probes labeled using the RadPrime DNA labeling system (Invitro-
gen). Probe details and primers used for amplification are listed in Tables 3
RESULTS AND DISCUSSION
When we first applied our established four-gene MLST
scheme (23) to additional diverse isolates from our collection,
we noticed the apparent, but unexpected and previously un-
recognized, relatedness of clinically important E. faecalis iso-
lates of diverse origins and resistance profiles. To further in-
vestigate the suggested relatedness, we used five additional
housekeeping genes (gki, gdh, aroE, xpt, and yqiL), represent-
ing altogether nine genes (loci) spread around the chromo-
some (28) (Fig. 1 and Table 2). A total of 5,287 bases, including
some results with the four genes from our previous study (23),
was sequenced from each of 21 selected isolates (Table 1).
Overall, we found 132 point mutations, two deletions, and 60
alleles (Fig. 2). The collagen adhesin gene ace, known to be
expressed during serious human E. faecalis infections (24),
showed greatest variability with 54 point mutations and one
69-bp in-frame deletion. Using the definitions described in
Materials and Methods, 14 STs consisting of six single isolate
STs, two clones (ST-2 and ST-12 are SLVs and ST-9 and ST-13
are DLVs), and one clonal complex (ST-6, ST-7, ST-14, and
ST-15) were identified among the 21 isolates (Table 1).
TABLE 2. Details of amplicons and primers used for nine-gene MLST
Gene (locus) Primer functionType Sequence (5?33?) Locations
ace (ef1099)PCR, sequencing
2,025 1,003 959
efaA (ef2076)927 735 693
salA (ef3060) 1,449962 922
pyrC (ef1718) 1,284360 320
gki (ef2788) 972 527483
gdh (ef1004) 1,524 528 484
aroE (ef1561)867 524480
xpt (ef2365)582 538494
yqiL (ef1364)2,412 523452
aFor one strain where we found mutations in the reverse primer of ace, this gene was amplified using an ace forward PCR primer (listed above) and a new reverse
primer with the sequence 5?-GCCATTCTCTTCAGTTGTTTCT-3?.
VOL. 187, 2005 EVOLUTION OF AN E. FAECALIS CLONAL COMPLEX AND PAI5711
The nine-gene MLST confirmed the relatedness of the pre-
viously studied (23) penicillinase-producing (?-lactamase,
Bla?) isolates of ST-6 and its SLV, ST-7 (Fig. 3A and Table 1).
The earliest isolate of this clone, HH-22, the first known Bla?
isolate of E. faecalis, is a multidrug-resistant urine isolate re-
covered in Texas in 1981 (18); the blaZ gene of this strain is
located on a plasmid that also harbors a gene for high-level
gentamicin resistance (HL-Gmr) (16). Other members of this
clone (which represents the majority of known Bla?isolates)
were subsequently found in five states of North America (20),
including a large, prolonged outbreak in a Virginia hospital
(33, 40) and a hospital outbreak that included five bloodstream
infections in North Carolina (13). Surprisingly, the first known
U.S. vancomycin-resistant enterococcus, E. faecalis strain V583
(32) (from Missouri), which has recently been sequenced (28),
was also found to be a member of this clonal cluster (ST-14),
differing from ST-6 by a single nucleotide in efaA. A Bla?,
vancomycin-susceptible, HL-GmrE. faecalis strain, MMH594,
representative of an outbreak (1984 to 1987) with increased
risk of death and isolated prior to V583 in a city from a nearby
state (8), also belongs to ST-14. Among several endocarditis
isolates tested, one (vancomycin susceptible, Bla?, and HL-
TABLE 3. Details of primers used for generating virulence-
associated gene probes
Locus (gene) TypeSequence (5?33?)
ef0080 (gls24) Forward
ef0818 (hylB) 841
ef1818 (gelE) Forward514
ef1821 (fsrB) Forward 500
aGene coding for Ig-like fold-containing putative surface adhesin (35).
bGene coding for putative lipase, an enzyme implicated in pathogenesis in
cAll genes were amplified from V583.
TABLE 4. Details of primers used for generating PAI-associated
ef0495 Forward 576
cbh (ef0521) Forward 508
araC (ef0530) Forward 547
ef0534 Forward 392
ef0539 Forward 548
xylA (ef0556) Forward562
aThe esp gene was amplified from strain MMH594, and all other genes were
amplified from V583.
5712NALLAPAREDDY ET AL.J. BACTERIOL.
Gmr) belongs to ST-15, differing from ST-14 by a single nu-
cleotide (pyrC); this isolate was recovered 6 years after V583
from another Missouri city.
This group of isolates, clustered as shown in Fig. 3A by
BURST analysis (9), was named the “BVE” (Bla?-Vanr-en-
docarditis) clonal complex; all members have a maximum dif-
ference of one allele from at least one other member. Because
of the overrepresentation of Bla?ST-6 isolates among isolates
tested, the central circle in Fig. 3A denotes the predominant
type and not the ancestral type (see below for predicted an-
cestral type). Scenarios to explain these results include the
possible existence of an ancestral lineage that spread and
evolved slowly over many years, until its disease-causing and
resistance acquisition potentials were recognized, or that the
group recently evolved into a more favorable form and then
spread rapidly, achieving distribution of a subpopulation in
various locations. This clonal cluster is notable because it has
not only demonstrated pathogenic potential (causing serious
infections in outbreaks [8, 13, 33, 40] as well as endocarditis)
but also has acquired two uncommon to rare (for E. faecalis)
resistances. Vancomycin resistance, seen predominantly in the
species E. faecium, is an uncommon property of E. faecalis,
found in ?2% of isolates, while Bla producers are even more
rare; of note, however, a vancomycin-resistant E. faecalis was
the donor of the vanA genes in at least one of the recent
descriptions of vancomycin-resistant methicillin-resistant S.
aureus (39). The BVE clone does not appear to be predomi-
nant among E. faecalis isolates in general and was found in
only 2 of over 70 other independent isolates examined, ana-
lyzed by two or more methods of PFGE, multilocus enzyme
electrophoresis (38), and MLST.
FIG. 1. Chromosomal locations of the nine MLST loci in the E.
faecalis V583 genome (28). The arrowheads inside the circle represent
the open reading frame orientations of each locus (not drawn to scale).
Antigenic gene arrowheads are shaded in black, and housekeeping
gene arrowheads are shaded in gray. The arrowhead of dnaA (coding
for chromosomal replication factor) positioned at nucleotide 1 is not
shaded. The distance between any two loci ranged from 103 kb to 1,247
kb. The putative PAI region is boxed.
FIG. 2. Allelic variation of the MLST loci sequenced for analysis. Allelic variations of ace, efaA, and salA were as previously published (23) and
hence are not shown here. For these three genes, alleles A to I of the previous study (23) were redefined as alleles 1 to 9 for this study. The
nucleotides present in each of the variable sites of allele 1 (E. faecalis OG1RF) are shown. Only those sites that differ are shown for the other
alleles. The position of each variable site within the sequenced fragment is shown by the number above the nucleotide, read vertically.
VOL. 187, 2005 EVOLUTION OF AN E. FAECALIS CLONAL COMPLEX AND PAI5713
Analysis of PFGE fingerprints of this BVE clonal complex
also showed related PFGE patterns, differing by only a few
fragments, which would categorize them as closely or possibly
related (Fig. 3B) using criteria for analysis of potential noso-
comial outbreaks (37). Hybridization of PFGE Southern blots
of chromosomal digestion fragments with probes for gdh, aroE,
yqiL, gki, and xpt showed hybridization to, for the majority of
fragments, the same-sized band in isolates of the BVE clonal
complex, with sizes as expected from the V583 genome. One of
the exceptions (a difference in size of the xpt hybridizing band
of V583) is known to be due to insertion of the vancomycin
resistance (vanB) element in this strain (28). Although PFGE
and hybridizations confirmed the relatedness of all the MLST-
defined BVE clonal complex isolates, the degrees of difference
inferred by the two techniques did not always strictly overlap;
this is not surprising, since PFGE and MLST have different
Among two other new relationships identified, one clone
named HV1 (Houston Vanr#1) represents the unrecognized
persistence of a clone identified earlier (1, 11, 23) in a single
hospital. Another unexpected observation was that two other
Bla?isolates (ST-9 and its DLV, ST-13, differing only in an-
tigenic genes), representative of small nosocomial outbreaks in
Connecticut (27) and in Argentina (17), respectively, belong to
the same clone, named ACB (Argentina-Connecticut-Bla?);
this clone is unrelated to the BVE clonal complex, with vari-
ations at eight of nine loci. Among other Bla?E. faecalis
isolates known to date, one isolate from Lebanon (20, 23, 38)
and an outbreak strain (20, 30) from Boston (not included in
this study) are not related to the BVE or ACB groups (20).
This implies that the staphylococcal blaZ gene has spread only
a few times into E. faecalis and suggests that certain back-
grounds may be particularly receptive to blaZ acquisition.
To further assess the genetic content of the isolates in this
study, we generated a profile based upon hybridization to 14
chromosomally encoded potential virulence genes (Fig. 4A and
Table 3). Many, including six recently described immunoglob-
ulin (Ig)-like fold-containing putative microbial surface ad-
hesins (35), were present in all 21 isolates. However, four genes
were variably present and the differences corresponded to dif-
ferent STs; the variably present genes (Fig. 4A) were fsrB
(encoding part of the Fsr two-component system of E. faecalis,
which regulates the virulence genes gelE and sprE ); ef1824
(encoding a predicted adhesin  with a characteristic Ig-like
fold ); and hylA and hylB (ef3023 and ef0818, respectively),
each encoding a putative hyaluronidase, an enzyme implicated
in pathogenesis in other organisms. Although gelE was found
in all 21 isolates, the lack of gelatinase production by isolates
in ST-4, ST-8, and ST-11 is related to a previously described
23.9-kb deletion (22) which includes fsrB. Based on the obser-
vation that ace B repeats (24) are separated by recer se-
quences, which may promote recombination and thus variable
repeat numbers (2), we also tested the number of B repeats of
the E. faecalis-specific collagen adhesin Ace (Fig. 4A). While
neither the gene profile nor ace B repeats were alone suffi-
ciently discriminatory, the combination of these two profiles
successfully distinguished the lineages from one another, re-
flective of the MLST and PFGE types (Fig. 4A). Notably, the
BVE clonal complex, including the two additional members
recognized by multilocus enzyme electrophoresis or PFGE
(both HL-Gmr) (38) but not tested by MLST, contained all of
the potential virulence genes.
Shankar et al. (34) recently proposed an ?150-kb region as
an E. faecalis PAI (ef0479 to ef0628 of V583) and indicated
that there were only subtle differences in this region in two of
the E. faecalis isolates described above, V583 (28, 32) and
MMH594 (8). However, our recognition that these two isolates
are actually members of the same ST suggests that the highly
FIG. 3. Analysis of an unusual E. faecalis clonal cluster by MLST and PFGE. (A) Clustering of four clonally related STs using BURST analysis.
The central circle denotes the predominant type among isolates tested using a nine-gene MLST scheme, and each surrounding circle indicates one
allele difference. A dashed straight line denotes a double locus difference. (B) PFGE fingerprints. The PFGE phylogenetic tree was based on the
unweighted pair group method. Tolerance of up to 5% shift in the band position was used. Isolates are generally referred to by their previously
published designations. Year and place of isolation and ST type are shown. Isolates from outbreaks are marked with an asterisk. V583 is the first
vancomycin-resistant enterococcus isolated in the United States, and HH-22 is the first-known Bla?isolate of E. faecalis. E228 and E366, isolated
a year apart from the same hospital and differing by three bands, are represented by E228.
5714 NALLAPAREDDY ET AL.J. BACTERIOL.
FIG. 4. Schematic presentation of hybridization profiles for potential virulence-related genes and PAI genes. (A) Virulence-related gene (non-PAI) profile and ace B
repeat number profile of 21 MLST-analyzed isolates. The 10 genes (of ef0080, ef0089, ef0786, ef1091, ef1092, ef1093, ef1269, ef1818, ef2224, and ef3191) present in all 21isolates are not shown. The STs with the 23.9-kb deletion involving fsrB are marked with a superscript “a” in the fsrB data. (B) Determination of the PAI insertion site by
PCR using primers within and outside the PAI of V583. The double arrow denotes an expected-size PCR fragment with primers PAIout plus ef0481forward or ef0482forward. STs yielding an ?1.7-kb-larger PCR product or an ?1-kb-larger PCR product are marked with superscript “b” and “c,” respectively. (C) Hybridization results with
PAI-associated intragenic probes, representing 18 genes dispersed over the entire PAI region. The superscript numbers on ST and the ?/? symbols denote the number of
isolates of that type. The integrated plasmid region (ef0506 to ef0485) (28) is boxed. (D) Distribution of three PAI-associated genes among 341 clinical isolates, 58 nosocomial stool isolates, 33 community-derived stool isolates, and 22 animal isolates.
VOL. 187, 2005 EVOLUTION OF AN E. FAECALIS CLONAL COMPLEX AND PAI5715
similar nature of the putative PAI of these isolates is a function
of their close evolutionary relationship. To further assess
whether a similar PAI was present in the other BVE clonal
complex isolates and in unrelated strains, we tested the 21
isolates described above, which belong to 14 STs within nine
different clonal lineages, with probes representing different
genes dispersed over the entire PAI region (Fig. 4C). Colony
lysate hybridization results with the 18 individually labeled
intragenic probes (Table 4) showed that all 18 genes (which are
all present in MMH594, in which the PAI was first identified
), are also present in two unrelated STs (ST-4 and ST-11,
both represented by HL-Gmrisolates from Thailand isolated
in 1980) (Fig. 4C). The remaining isolates representing 10 STs
contain an incomplete PAI with deletions in different regions,
except the ST-1 isolate (OGIRF ), which contains none of
the 18 genes (Fig. 4C). Hybridization of BVE clonal complex
members to xylA and gls24-like gene probes confirmed the
presence of these PAI genes within the same-sized PFGE
fragments of the BVE clonal complex. However, within this
complex, there were isolate-specific PAI deletions localized to
three regions, one including ef0530 and ef0534, a second in the
middle (ef0571), and the third including ef0604 and ef0609
(Fig. 4C). This finding of PAI variability is not unexpected,
considering the frequent occurrence of IS-like elements and
integrase and recombinase genes in the PAI region of
MMH594 and V583 (28, 34), including the previously de-
scribed 17-kb deletion in PAI of V583 versus MMH594, both
ST-14 isolates (34). Although there are many differences
within the PAI of isolates of different lineages, possibly due to
deletions, the finding of PAI-associated genes in eight of nine
FIG. 5. Predicted path of evolution of the BVE clonal complex based on MLST, deletions in the PAI region, year of isolation, and the presence
of uncommon resistance genes, blaZ and vanB. Isolates of the BVE clonal complex contained all 14 tested potential virulence genes (Fig. 4).
Isolates of each ST within the clonal complex are grouped by oval shading, and SLVs are denoted by overlapping shaded ovals. Arrows with
continuous and dashed lines represent the predicted and alternative evolutionary paths. Putative ancestral isolates are boxed with a dashed line.
Five partial deletions (?? to ??) of the PAI of E. faecalis strain MMH594, including that previously described for V583 (34), are shown. VanB,
vancomycin resistance encoded by the vanB gene.
5716 NALLAPAREDDY ET AL.J. BACTERIOL.
lineages containing isolates from around the world corrobo-
rates the earlier proposal that PAI is disseminated among
To investigate a possible common insertion site of appar-
ently transferable PAI (25) in isolates from different STs, PCR
was performed using one primer located outside the PAI
(PAIout) and the second located within the PAI region
(ef0481forward or ef0482forward [Table 4]); two isolates lack-
ing both ef0481 and ef0482 were not tested. Products of 3.8 kb
(with ef0481forward primer) and 4.2 kb (with ef0482forward
primer) (sizes were as anticipated from the V583 genome
sequence) were obtained with DNA from 16 of 19 isolates (Fig.
4B), suggesting the same PAI insertion site in different E.
faecalis clones. Using the same sets of primers, ?1.7-kb and
?1-kb larger PCR products were obtained with DNA from the
HV1 clone and from the ST-8 isolate, respectively, indicating
further small insertions in this region. Thus, these results pre-
dict that at least seven of the nine lineages have a common PAI
We also tested an additional 454 geographically and tempo-
rally diverse isolates for the presence of three selected PAI-
associated genes, one close to each end and one in the middle.
Hybridization results indicate that all three PAI-associated
genes (esp, xylA, and gls24-like) are distributed worldwide and
are enriched in infection-derived isolates (P ? 0.0025 com-
pared to community-derived isolates from human stools or
animals), extending a previous report using 80 isolates of un-
known clonal relatedness (34). Among the 341 clinical isolates,
17.6% were found to contain all three genes, 41.4% contained
combinations of esp plus xylA or xylA plus gls24-like, and 27%
of isolates lacked all three genes. The variability of the PAI
region is consistent with the many deletions identified above
for the well-characterized lineages (Fig. 4D). The frequent
finding of two or more PAI genes, together with the results for
a common insertion site, suggests that these PAI genes were
acquired as a unit with subsequent deletions. The less frequent
occurrence of PAI-associated genes in nosocomial stool iso-
lates compared to nosocomial clinical isolates (49.9% versus
31% for esp; 62.5% versus 39.7% for xylA) is likely because
fecal isolates of hospitalized patients include a mixture of both
nosocomially derived and community-derived organisms. The
uncommon occurrence of even one of these three PAI-associ-
ated genes in non-human-derived isolates (9.1%) plus the high
frequency of occurrence of at least one of the three genes in
clinical isolates (73%) support the hypothesis that this genomic
region may be helpful during some stage of human infection.
The results also suggested that some deletions may be favored,
or may be a clonal marker, in specific clinical settings, as
exemplified by the very frequent absence of the gls24-like re-
gion in endocarditis isolates.
In a further analysis of individual isolates of the unusual
BVE clonal complex, we used the PAI region variability, to-
gether with locus variations and the presence of antibiotic
resistances, to predict the evolutionary pathway of this distinc-
tive lineage (Fig. 5). The most complete PAI region (like that
present in two Thailand strains) was found in MMH594, which
lacks blaZ and vanB, and so this isolate or some predecessor
was positioned in an ancestral position. A large PAI region
(including cylM, the 17-kb region previously noted as deleted
from V583 , araC [ef0530], and ef0534) was missing from
all Bla?(ST-6 and ST-7) isolates of this clonal complex (Fig.
4C and 5, ?B), and long-range PCR with primers outside this
deletion (ef0521reverse and ef0539forward [Table 4]) yielded
the same-sized PCR fragments (?10 kb) with all Bla?isolates
of the BVE clonal complex, confirming the same deletion in
these isolates. An additional region (ef0604 to ef0628) (Fig. 5,
?E) of the PAI was absent in a single ST-7 isolate, HH-22,
suggesting that this isolate, although it was the first Bla?en-
terococcus identified, was not the ancestor of the later, more
widespread ST-6 isolates. The endocarditis isolate of the BVE
clonal complex showed two independent PAI deletions, one in
the middle (ef0571) and the other including ef0604 and ef0609
(Fig. 4C and 5, ?C and ?D). In our scheme, the acquisition of
HL-Gmris inferred to have occurred after PAI acquisition,
although the reverse could also be true.
In summary, we have identified and characterized a unique
E. faecalis clonal complex which can cause outbreaks and life-
threatening infections and has acquired HL-Gmras well as, at
different times, ?-lactamase and vancomycin resistance, two
unusual resistances for this species. These three resistances
eliminate the activity of the cell wall-active agents most com-
monly used for E. faecalis infections, ampicillin and vancomy-
cin, and of gentamicin, the aminoglycoside most often used for
synergism when treating enterococcal endocarditis. Height-
ened awareness and the ensuing study of this unusual clonal
complex may lead to improved understanding of its incidence,
pathogenicity, clinical associations, and evolving patterns of
antimicrobial resistances and thus may provide valuable infor-
mation for control of spread and human disease caused by E.
We acknowledge the many physicians and researchers around the
world for providing isolates for our 30-year strain collection. We thank
Kavindra V. Singh for his help and Karen Jacques-Palaz for her tech-
This work is supported by NIH grant R37 AI47923 from the Division
of Microbiology and Infectious Diseases to B. E. Murray.
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