APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2007, p. 3307–3319
0099-2240/07/$08.00⫹0 doi:10.1128/AEM.02239-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 10
VanA-Type Enterococci from Humans, Animals, and Food: Species
Distribution, Population Structure, Tn1546 Typing and Location,
and Virulence Determinants䌤
F. Biavasco,1* G. Foglia,1 C. Paoletti,1 G. Zandri,1 G. Magi,1 E. Guaglianone,2 A. Sundsfjord,3
C. Pruzzo,4 G. Donelli,2 and B. Facinelli1
Institute of Microbiology and Biomedical Sciences, Polytechnic University of Marche, Ancona, Italy1; Department of
Technologies and Health, Istituto Superiore di Sanità, Rome, Italy2; Department of Microbiology and Virology,
University of Tromsø, Tromsø, Norway3; and Department of Biology, University of Genoa, Genoa, Italy4
VanA-type human (n ⴝ 69), animal (n ⴝ 49), and food (n ⴝ 36) glycopeptide-resistant enterococci (GRE)
from different geographic areas were investigated to study their possible reservoirs and transmission routes.
Pulsed-field gel electrophoresis (PFGE) revealed two small genetically related clusters, M39 (n ⴝ 4) and M49
(n ⴝ 13), representing Enterococcus faecium isolates from animal and human feces and from clinical and fecal
human samples. Multilocus sequence typing showed that both belonged to the epidemic lineage of CC17. purK
allele analysis of 28 selected isolates revealed that type 1 was prevalent in human strains (8/11) and types 6 and
3 (14/15) were prevalent in poultry (animals and meat). One hundred and five of the 154 VanA GRE isolates,
encompassing different species, origins, and PFGE types, were examined for Tn1546 type and location (plasmid
or chromosome) and the incidence of virulence determinants. Hybridization of S1- and I-CeuI-digested total
DNA revealed a plasmid location in 98% of the isolates. Human intestinal and animal E. faecium isolates bore
large (>150 kb) vanA plasmids. Results of PCR-restriction fragment length polymorphism and sequencing
showed the presence of prototype Tn1546 in 80% of strains and the G-to-T mutation at position 8234 in three
human intestinal and two pork E. faecium isolates. There were no significant associations (P > 0.5) between
Tn1546 type and GRE source or enterococcal species. Virulence determinants were detected in all reservoirs
but were significantly more frequent (P < 0.02) among clinical strains. Multiple determinants were found in
clinical and meat Enterococcus faecalis isolates. The presence of indistinguishable vanA elements (mostly
plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence
of host-specific purK housekeeping genes suggested that all GRE might be potential reservoirs of resistance
determinants and virulence traits transferable to human-adapted clusters.
Enterococci are gram-positive, opportunistic bacteria that inhabit the gastrointestinal tracts of humans and many animals.
They are also present in food, as starter cultures for the production of cheese and fermented sausages or as fecal contaminants of
raw meat, milk, and milk products. Some specific strains are
available as probiotics in animal feeds (20, 22). However, enterococci have gained notoriety as a major cause of nosocomial infection and are increasingly isolated from the bloodstream, urinary tract, and surgical sites. Enterococcus faecalis causes 80 to
90% of human enterococcal infections, and Enterococcus faecium
causes most of the remaining cases (other enterococcal species
being infrequently involved) (27). The emergence of multidrug
resistance (i.e., resistance to multiple antibacterial agents), including high-level resistance to glycopeptides, among enterococci,
particularly E. faecium, has resulted in clinical isolates resistant to
all antibiotics of proven efficacy (7). Glycopeptide-resistant enterococci (GRE), which have emerged as nosocomial pathogens
in the past 10 to 15 years (7), are a global problem despite major
epidemiological differences between Europe and the United
States. A high frequency of GRE has been reported in hospitals
in the United States, whereas extremely low frequencies have
been reported in the community, in animals, and in food of
animal origin (7, 12, 29, 39, 51).
Results of studies conducted in northern European countries
have revealed a low overall prevalence of GRE infection in Europe, with GRE being detected mostly in the nonhospitalized
healthy population and among animals (7, 51). However, their
incidence in clinical infections has been rising in southern Europe
(Portugal, Greece, and Italy) and, though remaining generally
lower that those described in the United States (European Antimicrobial Resistance Surveillance System, http://www.earss.rivm
.nl, last accessed 10 October 2005), rates of 20% for clinical
infections and of 7.5% for intestinal colonization in at-risk hospital wards have been reported in Italy (25).
The spread of GRE was linked to the use of the glycopeptide
avoparcin as a growth promoter in animal husbandry (37, 51),
until its ban in the European Union in 1997. Despite indirect
evidence for dissemination to humans of glycopeptide resistance selected in animals by clonal spread or horizontal resistance gene transfer (51), in Italy and other southern European
countries GRE isolation has so far been reported mostly in
hospital settings (7, 25; http://www.earss.rivm.nl), whereas relatively few data are available with regard to isolation in the
community and nonhuman sources (5, 7, 15).
* Corresponding author. Mailing address: Institute of Microbiology
and Biomedical Sciences, Polytechnic University of Marche, Via
Ranieri, Monte d’Ago, 60131 Ancona, Italy. Phone: 39 071 2204 637.
Fax: 39 071 2204 622. E-mail: f.biavasco@univpm.it.
䌤
Published ahead of print on 9 March 2007.
3307
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Received 22 September 2006/Accepted 1 March 2007
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BIAVASCO ET AL.
APPL. ENVIRON. MICROBIOL.
TABLE 1. Strains collected in this study
Strain and origin (n)
Italian strains
Human intestinal (43)
Animal (43)
Inpatientsa (37)
Area
North
1997
2002
Center
1997
2002
Outpatients (6)
North
Center
2002
2002
2003
Poultry (38)
North
1997
Pig (5)
Center
Center
2001
2003
2003
Poultry meat (27)
North
Center
Pork meat (7)
Human clinical (21)
Cheese (2)
Center
South
North
Blood (10)
North
PD1, VI2, PD3, VI4, VR5, PD6, PD7, VR8, VI9, VI10, VI11,
VR12, PD13, PD33, PD34, PD35, PD36, PD37, PD38,
PD39, PD40, PD41, PD42
VE31, VE44, VE45, VE46, VE47, VE50
AN14, AN25, AN26, AN27, AN28, AN29, AN30, AN43, AN49
AN21, AN22, AN23, AN24, PG15
2001
1998
2003
2003
1998
2003
PM3, PM12, PM13
PM14, PM15, PM16, PM17, PM18, PM19, PM20, PM21,
PM22, PM23, PM24, PM25, PM31, PM32
PM26, PM27, PM28, PM33
PM10, PM11
PM2, PM30, PM35, PM36
KM1
KM4, KM5, KM6, KM7, KM8, KM9
CH29, CH34
VI4, MI66
MI65
R35, AN64
R36, R39, R40
AN113, AN338
VI2, VI3, UD5
GE69
AN21, AN67
AN22
R38, AN68
R37
UD6
Center
North
Community
Norway
1998
HI-N41 to HI-N45 (31)
Poultry
Norway
Belgium
1998
1997
A-N16 to A-N20 (31)
FAIR-E-16210 (EU-FAIR project CT97-3078)
North
Center
a
VI1, VI46
MI24, MI25, MI26, MI28, MI29, MI30, MI31, MI32, MI33,
MI34, MI54, MI55, MI56, MI57, MI58, MI59, MI60
AN8
AN7, AN9, AN12, AN13, AN14, AN15, AN16, AN17, AN19,
AN20, AN23, AN47, AN48, AN49, AN50, AN51, AN52
MI27
AN10, AN11, AN18
AN53, AN61
Wound (1)
Bile (1)
Urine (9)
Animal (6)
1998
2000
Isolate(s)
1997
1998
1998
2002
2003
1997
2001
1997
1998
2002
2002
1997
Center
Non-Italian strains
Human intestinal (5)
Yr
No enterococcal infection.
The VanA phenotype, expressing inducible, high-level vancomycin and teicoplanin resistance, is the most common in
Europe (3, 5, 25, 29, 37). The vanA cluster—detected primarily
in E. faecium and E. faecalis and less frequently in other enterococcal species—is carried by Tn1546 and is transferable by
conjugation (2, 9). Considerable heterogeneity may exist
among Tn1546 elements, largely resulting from the presence of
insertion sequences or from deletions in nonessential genes
and intergenic regions (32, 51, 57).
The pathogenesis of enterococcal infections is still poorly
understood, although several virulence factors, such as aggregation substance(s) (AS), gelatinase (Gel), cytolysin
(Cyl), and enterococcal surface protein (Esp), have been
described (24, 27). AS are pheromone-inducible surface
proteins of E. faecalis that facilitate the conjugative ex-
change of plasmids (carrying virulence and/or antibiotic resistance genes) and also contribute to pathogenicity by enhancing adhesion to and internalization by cultured human
cells, as well as favoring intracellular survival within macrophages (10, 52, 55). Although sex pheromone plasmids are
highly specific for E. faecalis, they have also been detected in
vancomycin-resistant E. faecium strains (28, 40). Gel, a secreted Zn metalloprotease, and Cyl, a lytic toxin, have been
implicated in the pathogenicity of E. faecalis on the basis of
both epidemiological data and studies of animal models (24,
27, 48). Esp is a surface protein involved in the ability to
colonize and in immune evasion in E. faecalis and E. faecium
(21, 24). Enterococci are also known to produce slime (17,
18) and to form biofilms, which have been regarded as
virulence features of clinical isolates (16, 18).
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Food (36)
Source (n)
VOL. 73, 2007
TYPING AND VIRULENCE OF vanA ENTEROCOCCI
3309
TABLE 2. Control strains used in the study
Strain
Purpose
E. faecium BM4147
E.
E.
E.
E.
E.
E.
E.
E.
faecalis V583
gallinarum ATCC 49573
casseliflavus ATCC 14432
durans ATCC 19432
faecalis OG1RF(pCF10)
faecalis OG1RF(pAD1)
faecalis JH2-2
faecalis OG1RF
Glycopeptide resistance genotype, Tn1546 typing, probe synthesis and ddl PCR; vanA
control strain
Glycopeptide resistance genotype, ddl PCR; vanB control strain
Glycopeptide resistance genotype, ddl PCR; vanC-1 control strain
Glycopeptide resistance genotype, ddl PCR; vanC-2 control strain
ddl PCR
Clumping, aggregation substance identification; sex pheromone plasmid-harboring strain
Clumping, aggregation substance identification; sex pheromone plasmid-harboring strain
Sex pheromone production
Sex pheromone production
MATERIALS AND METHODS
Bacterial strains and media. We studied a total of 154 VanA-type GRE from
humans (n ⫽ 69), animals (n ⫽ 49), and food (n ⫽ 36). See Table 1 for details.
Most of the strains (n ⫽ 142) were collected throughout Italy between 1997 and
2003 (64 human, 42 animal, and 36 food isolates). The 11 non-Italian GRE
isolates were from Belgium (n ⫽ 1) and Norway (n ⫽ 10); of these, five were
human intestinal and six were animal isolates. Reference and control strains are
listed in Table 2.
GRE were isolated on Slanetz-Bartley agar (Becton Dickinson, Milan, Italy)
containing 6 g/ml vancomycin. Fecal and meat homogenates were previously
enriched in selective tryptone soy broth (Oxoid, Basingstoke, United Kingdom)
containing 0.4 mg/ml sodium azide and 6 g/ml vancomycin. Brain heart infusion
broth and agar (Oxoid) were used for routine culture. Tryptone soy broth
supplemented with 1% glucose was used for slime production assays. Gelatin
infusion broth containing 40 mg/ml gelatin (Bio-Rad Laboratories, Richmond,
CA) was used to determine gelatinase production. Blood agar base (Oxoid)
supplemented with fresh horse blood (5%) was used to investigate hemolysin
production. Isolates were maintained in glycerol at ⫺70°C and subcultured twice
on Slanetz-Bartley agar before testing.
Species identification and PFGE typing. GRE were identified at the species
level with API ID32-STREP kits (bioMérieux Italia, Rome, Italy) and additional
biochemical tests (22) and species-specific enterococcal ddl PCR performed with
the primers listed in Table 3. PFGE of SmaI (New England Biolabs, Beverly,
MA)-digested total DNA was performed essentially as described previously (3)
using a CHEF Mapper apparatus (Bio-Rad) with pulse time increasing from 1 to
20 s over 20 h at 200 V (6 V/cm). Genetic relatedness was interpreted according
to the method of Tenover et al. (53). Strains differing by six or fewer bands were
grouped into the same PFGE type (1, 2, 3, etc.) and subdivided into PFGE
subtypes (1a, 1b, 1c, etc.) based on single-band differences. PFGE data were
analyzed separately for each species, considering each band as a separate putative locus and scoring it as present (1) or absent (0) in each accession. Dendro-
38
45
ATCC
ATCC
ATCC
10
10
40
40
grams were constructed by the use of the Dice coefficient and the unweightedpair group method using arithmetic averages.
MLST. MLST was performed as described by Homan et al. (30) and included
the genes purK, adk, atpA, ddl, gdh, gyd, and pstS. Sequence types (ST) were
obtained from the MLST database at http://www.mlst.net. PCR was performed in
a Gene Amp PCR system 2400 thermal cycler (Applied Biosystems, Foster City,
CA). Amplification reactions were carried out in a 50-l final volume containing
2.5 U AmpliTaq Gold (Applied Biosystems). Amplified fragments were purified
from the reaction mix using a Montage PCR purification kit (Millipore Corporation, Bedford, MA). Sequencing was performed using ABI Prism (Applied
Biosystems) with dye-labeled terminators; sequences were analyzed by the
ClustalW method available at http://align.genome.jp.
Detection of glycopeptide resistance and virulence genes. Total DNA extraction was done as described previously (3). Vancomycin resistance and virulence
genes were detected by PCR using a Hybaid PCR Express thermal cycler
(Hybaid Ltd, Ashford, United Kingdom). Primers and target genes are listed in
Table 3. Virulence genes were detected using primers internal to (i) highly
conserved regions in the AS genes of pAD1, pPD1, and pCF10 of E. faecalis, (ii)
asa373 of pAM373, (iii) gelE, (iv) cylB, and (v) esp. EcoRI digestion of AGG
amplicons was performed according to the manufacturer’s instructions (Roche
Molecular Biochemicals, Mannheim, Germany), and restriction fragments were
separated by 2.0% agarose gel electrophoresis.
vanA gene location. The plasmid or chromosomal location of vanA was assessed using three different methods: (i) vanA hybridization of plasmid content
extracted by an alkaline lysis method; (ii) vanA hybridization of S1-digested total
DNA; (iii) and vanA hybridization of I-CeuI-digested total DNA. The first two
methods were performed as described previously (23) using a biotin-labeled
vanA probe and a BrightStar BioDetect kit (Ambion, Huntingdon, United Kingdom). vanA hybridization of I-CeuI-digested total DNA was performed essentially as described previously (33). DNA was digested with 5 U of I-CeuI (New
England Biolabs), separated by PFGE, transferred onto a nylon membrane, and
hybridized sequentially with 16S rRNA gene and vanA biotin-labeled DNA
probes.
Molecular analysis of Tn1546-like elements. The structure of Tn1546-like
elements was analyzed by PCR and amplicon restriction analysis (PCR- restriction fragment length polymorphism) essentially as described by Palepou et al.
(43). Long PCR was performed using TaKaRa Ex Taq (Cambrex Bio Science,
Milan, Italy) and a Hybaid PCR Express thermal cycler. Primers (sequence and
position) and target genes are listed in Table 3. Long PCR amplicons of the
whole Tn1546 were analyzed by digestion with ClaI, whereas amplicons of the
orf2-vanX region were digested with DdeI to detect the point mutation at position 8234 (31). Strains giving different results from the prototype were amplified
using primer pairs targeting the left (orf1-orf2) and right (vanX-vanZ) region of
Tn1546.
Phenotypic assays. Clumping assays were performed as described previously
(40). Production of Gel was determined as described previously (11). For -hemolysis detection, strains were grown on horse blood agar plates for 1 to 2 days
at 37°C. Biofilm formation was tested using the slime production assay described
previously (17).
Statistical analysis. The prevalence of the different species, different Tn1546
types, and virulence traits in the various reservoirs were compared using Fisher’s
test. Statistical analysis was performed with the S-PLUS 6 statistical program
(S-PLUS 6.1 for Windows, Professional Edition, release 1). A P of ⬍0.05 was
regarded as statistically significant.
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Several reviews have addressed the genetic basis, reservoirs,
and spread of glycopeptide resistance in enterococci (7, 9, 51)
and enterococcal virulence (24, 27, 47). A combination of glycopeptide resistance and virulence in enterococci could pose a
serious threat to human health. However, data on the presence
of virulence traits in GRE are scarce (42, 44).
The present study was undertaken to explore the relatedness of GRE of different origins (human, animal, and food)
and from different geographic areas to gain a better understanding of the involvement of the different reservoirs in the
emergence and spread of virulent clones, i.e., those that in
addition to antibiotic resistance have also acquired a number of genes conferring infectivity and virulence. To do this,
human, animal, and food GRE were analyzed for population
structure (using pulsed-field gel electrophoresis [PFGE],
purK allele sequence analysis, and multilocus sequence typing [MLST]) and Tn1546 type and location (chromosome or
plasmid) as well as for the presence and expression of the
main virulence determinants.
Reference or source
3310
BIAVASCO ET AL.
APPL. ENVIRON. MICROBIOL.
TABLE 3. PCR primers and products
Purpose and primers
Gene target(s)
Product
length (bp)
Positiona
Primer sequence (5⬘–3⬘)
Reference
485–506b
868–845b
485–506b
649–672b
359–377
887–870
98–116
1038–1021
39–58b
240–260b
964–944b
378
36
189
36
528
19
941
19
957
726
35
14
14
Glycopeptide resistance
genotype
VANA1
VANA2
VANB
VANB2
VANC1-1
VANC1-2
VANC2/3-1
VANC2/3-2
175–191
907–891
173–189
807–791
246–272
1067–1051
455–486
885–869
732
19
635
19
822
19
439
19
Tn1546
IR
INV2
VANX2
ORF1A
ORF1B
ORF1C
ORF2R
INV3
VANZ1
Virulence factors
AGG1
AGG2
ASA373F
ASA373R
GELE1
GELE2
CYLB1
CYLB2
ESP1
ESP2
a
b
c
vanA
vanB
vanC1
vanC2/3
Tn1546
orf2-vanX
orf1
orf1
orf1
orf2
vanX-vanZ
prgB, asaI, aspI
asa373
gelE
cylB
esp
GGGAAAACGACAATTGC
GTACAATGCGGCCGTTA
ATGGGAAGCCGATAGTC
GATTTGCTTCCTCGACC
GGTATCAAGGAAACCTC
CTTCCGCCATCATAGCT
CTCCTACGATTCTCTTG
CGAGCAAGACCTTTAAG
GGAAAATGCGGATTTACAACGCTAAG
13–38, 10814–10839
ATGAGGTGATATTTTGCGGAAA
3174–3195*
CTATTGGGGTATGGTTCGTCT
8579–8599*
AGGGCGACATATGGTGTAACA
170–190*
TGGTGGCTCCTTTTCCCAGTTC
907–928*
ACCGTTTTTGCAGTAAGTCTAAAT
1871–1894*
TTTCCGCAAAATATCACCTCAT
3195–3174*
AGACGAACCATACCCCAATAG
8578–8596*
GGTACGGTAAACGAGCAATAATA
10577–10555*
AAGAAAAAGAAGTAGACCAAC
AAACGGCAAGACAAGTAAATA
GGACGCACGTACACAAAGCTAC
CTGGGTGTGATTCCGCTGTTA
ACGCATTGCTTTTCCATC
ACCCCGTATCATTGGTTT
ATTCCTACCTATGTTCTGTTA
AATAAACTCTTCTTTTCCAAC
TTGCTAATGCTAGTCCACGACC
GCGTCAACACTTGCATTGCCGA
601–622
2156–2133
3094–3115
3693–3713
729–746
1148–1129
1199–1219
2041–2021
1217–1238
2149–2128
10,826
5,405
1,999
43
23
4
41
41
41
This study
23
4
1,555
20
619
13
419
20
843
20
932
20
Positions are from the first base of the coding sequence, except for positions marked with asterisks, which are from the first base of IRL of Tn1546.
Escherichia coli numbering.
The P1-16S2 pair was used for species identification; the 16S1-16S2 pair was used for probe synthesis.
RESULTS
Species and glycopeptide resistance genotype identification.
Of the 154 GRE, there were 120 E. faecium, 18 E. durans, 12
E. faecalis, and 4 E. gallinarum. Species prevalence in the
different reservoirs is reported in Table 4. As expected, E.
faecium was always the prevalent species (P ⬍ 0.02), followed
by E. faecalis (among human specimens) and by E. durans
(among nonhuman samples). E. faecalis was not found among
animal nor E. durans among human samples. E. faecalis was
more frequently recovered from the clinical than the other
reservoirs (P ⬍ 0.05). Among the other reservoirs (i.e., animal,
food, and human intestinal), the sole significant difference in
its frequency was between food and animal samples (P ⫽ 0.03).
The results of multiplex PCR targeting vanA, vanB, and
vanC showed that all isolates carried the vanA gene, including
the four vanC-1 E. gallinarum.
PFGE typing. All isolates were PFGE typed after SmaI
digestion of total DNA, yielding 69 different PFGE types (E.
TABLE 4. Species distribution in the different reservoirs
No. of isolates
Origin
Human intestinal
Animal
Food
Human clinical
E. faecium
E. faecalis
E. durans
E. gallinarum
47
35
24
14
1
0
4
7
0
12
6
0
0
2
2
0
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Identification and 16S probe
HiF
ddlE. hirae
TTATGTCCCTGTTTTGAAAAA
HiR
TTTTGATAGACCTCTTCCGGT
DuF
ddlE. durans
TTATGTCCCAGTATTGAAAAA
DuR
TGAATCATATTGGTATGCAGT
DDLM1
ddlE. faecium
TAGAGACATTGAATATGCC
DDLM2
CTAACATCGTGTAAGCT
DDLS1
ddlE. faecalis
ATCAAGTACAGTTAGTCT
DDLS2
ACGATTCAAAGCTAACTG
P1
rRNA gene 16S GCGGCGTGCCTAATACATGC
16S1
TGCATTAGCTAGTTGGTGAGG
16S2c
TCGAATTAAACCACATGCTCC
VOL. 73, 2007
TYPING AND VIRULENCE OF vanA ENTEROCOCCI
3311
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FIG. 1. Dendrograms showing the similarity index among the 154 isolates of E. faecium (A) and E. faecalis, E. durans, and E. gallinarum (B).
Clusters sharing ⱖ70% similarity are shown in gray. A, animal isolate; F, food isolate; HC, human clinical isolate; HI, human intestinal isolate.
CI, central Italy; NI, northern Italy; SI, southern Italy; NO, Norway; BE, Belgium. PFGE types showing a clonal spread are boxed.
faecium M1 to M49, E. faecalis S1 to S10, E. durans D1 to D7,
and E. gallinarum G1 to G3) and 30 PFGE subtypes. Results
are represented in four dendrograms, one per species (Fig. 1).
Ten clusters of E. faecium, one of E. faecalis, and three of E.
durans were evidenced. Two E. faecium PFGE types (M39 and
M49) provided evidence of clonal spread; type M39 (n ⫽ 15)
was isolated from both human (n ⫽ 12, in- and outpatients)
and animal (n ⫽ 3, pig) samples, whereas type M49 (n ⫽ 26)
3312
BIAVASCO ET AL.
APPL. ENVIRON. MICROBIOL.
TABLE 5. purK allele in 28 strains of different origins
and PFGE types
Origin and strain
Source
PFGE type
purK
allele
Inpatient
Inpatient
Inpatient
Inpatient
Community
Community
Community
M48
M49g
M34
M49l
M39
M21
M27
1
1
1
1
1
6
6
Animal
A-PD1
A-VI2
A-VI10
A-VR12
A-N18
A-N19
A-AN26
A-VE31
A-PD33
A-PD35
A-AN23
Poultry
Poultry
Poultry
Poultry
Poultry
Poultry
Poultry
Poultry
Poultry
Poultry
Pig
M25
M22b
M18
M32
M29a
M37
M2
M4
M20
M38
M39
2
3
3
3
6
6
6
6
6
6
1
Food
F-PM11
F-PM12
F-PM19
F-PM22
F-PM30
F-KM9
Poultry
Poultry
Poultry
Poultry
Poultry
Pork
M16
M45
M13a
M9
M42
M10
3
6
6
6
6
9
Human clinical
HC-AN64
HC-BG65
HC-R38
HC-AN67
Blood
Blood
Urine
Urine
M24
M23
M49n
M11
1
1
1
3
was isolated from inpatients (both fecal [n ⫽ 19] and clinical
[n ⫽ 7] samples). Six additional PFGE types were recovered
from different sources: M7, D1, D5, and G2 from both animals
and food, M29 from both human intestine and animals, and
M42 from both human clinical and food (meat and cheese)
samples. Type M30 was detected in fecal samples from both inand outpatients.
With regard to geographic spread, type M7 was recovered
from two isolates, one from Italy (poultry meat) and one from
Norway (chicken feces), type D1 was collected throughout
Italy, type D5 in northern Italy, types M39, G2, and M30 in
central Italy, type M29 in Norway, and types M49 and M42 in
central and northern Italy.
purK allele analysis. purK allele polymorphisms were determined in 28 isolates of different origins (5 clinical, 6 human
intestinal, 11 animal, and 6 food) and PFGE types. Five purK
alleles were found, with types 1, 6, and 3 being detected in
multiple strains (Table 5). Type 1 was found mostly in human
strains (3/5 clinical and 5/6 intestinal), type 6 in poultry (6/10
animal and 4/5 meat), and type 3 in poultry (3/10 animal).
MLST. To gain a better understanding of the clonal lineage
of the two major PFGE clusters, M39 and M49, MLST was
performed on five isolates representing different reservoirs,
two from cluster M39 (HI-AN10 and A-AN23) and three from
Downloaded from http://aem.asm.org/ on February 16, 2016 by guest
Human intestinal
HI-VI1
HI-MI28
HI-MI29
HI-AN47
HI-AN10
HI-MI27
HI-N44
M49 (HI-MI28, HI-AN47, and HC-R38, subtype M49g, M49l,
and M49n, respectively). ST 18 was found in the two M39
isolates (one from outpatient feces and one from a pig), and ST
78 was found in the three M49 isolates (two from inpatient
feces and one clinical).
One hundred and five isolates (9 E. faecalis, 78 E. faecium,
14 E. durans, and 4 E. gallinarum strains), encompassing different origins and PFGE types, were selected for further studies, i.e., vanA gene location, molecular analysis of Tn1546
elements, and virulence traits.
vanA gene location. Hybridization of plasmid content following alkaline lysis extraction demonstrated a plasmid location of
vanA in 77 of the 105 strains (Fig. 2). Fifty-four isolates, including the 28 that did not hybridize and 26 isolates showing a
positive reaction, were subjected to vanA hybridization of S1digested total DNA, which allows better identification of highmolecular-weight plasmids. A plasmid location of vanA was
demonstrated with this method in 24 of the previously negative
28 isolates and was confirmed in all of the 26 positive ones. The
size of the vanA-carrying plasmids ranged from 150 kb to 250
kb in the former and from 25 kb to 150 kb in the latter isolates.
The same 54 isolates were then analyzed by sequential hybridization of I-CeuI-digested total DNA with 16S rRNA gene and
vanA probes. All tested strains hybridized with the 16S rRNA
gene probe, and seven strains (E. faecium HI-MI30, HI-MI25,
HI-MI31, HI-MI32, and HI-MI60 and E. gallinarum A-BE48
and F-PM3) also hybridized with the vanA probe, demonstrating a chromosomal location of the vanA gene. In S1 digestion
experiments, none of the E. gallinarum isolates hybridized with
the vanA probe, whereas in all of the five E. faecium isolates,
vanA was detected on a 240-kb plasmid, thus demonstrating
the presence of two copies of vanA (Fig. 3). In E. faecium
HI-AN20 and HI-AN15, the vanA location could not be assessed with any experimental approach.
Molecular analysis of Tn1546-like elements. The 105 vanA
isolates were analyzed for the structure of the Tn1546 element
and assigned to 12 different groups (Fig. 4). Overall, PCR
experiments with primer IR gave a positive result in 93 isolates.
In 83 isolates, they yielded a single amplicon identical in size to
the prototype Tn1546 element, as also confirmed by ClaI restriction analysis; in six isolates (HI-VI1, HI-MI29, HC-AN64,
HC-VI2, F-KM5, and A-PD3), they gave rise to an amplicon
larger than the prototype; and in the remaining four isolates
(HI-MI25, HI-MI31, HI-MI32, and HI-MI60), they yielded
two amplicons, one of the same size as the prototype and one
larger.
The 10 isolates differing from the prototype and the 12
isolates not yielding amplicons were further studied by PCR. In
the six isolates harboring a single Tn1546-like element larger
than the prototype, the ORF1A-ORF2R primer pair yielded
an amplicon corresponding to the prototype in four cases
(E. durans F-KM5, E. faecium HI-VI1, E. faecium HC-VI2,
and E. faecium HI-MI29), an amplicon larger by 900 bp in one
(E. durans A-PD3), and no amplicon in the remaining isolate
(E. faecium HC-AN64); the INV3-VANZ1 pair yielded an
amplicon of the expected size in E. durans A-PD3 and E.
durans F-KM5 (suggesting an insertion downstream of
VANZ1 in this strain) and an amplicon larger by 800 bp to
1,900 bp in the remaining isolates (Fig. 4). The four isolates
with two IR amplicons, one corresponding to and one larger
VOL. 73, 2007
TYPING AND VIRULENCE OF vanA ENTEROCOCCI
3313
than the prototype, all gave the same results, i.e., ClaI digestion yielded three extra fragments, suggesting the presence of
a Tn1546 element 3 kb larger than the prototype. Tn1546
amplification using ORF1A-ORF2R and INV3-VANZ1, targeting the ends of the transposon, and further PCR experiments using INV2-VANX1, targeting the core region, yielded
amplicons comparable in size to those of the prototype, indicating the presence of insertions downstream of VANZ1 or
upstream of ORF1A (data not shown).
Primer pair INV3-VANZ1 yielded an amplicon corresponding to the prototype in all 12 isolates giving no IR amplicons,
indicating a prototype vanA cluster on the right end, whereas
ORF1A-ORF2R yielded an amplicon of the expected size in
only two isolates (E. faecium F-KM8 and HI-AN18), arguing
for the presence in these strains of deletions upstream of nucleotide 170 (Fig. 4). Additional PCR experiments were performed to establish the size of the left-end deletion in the
remaining 10 isolates. Primer pair ORF1B-ORF2R yielded a
product corresponding to the prototype in one strain (E. faecium A-AN26), suggesting a left-side deletion until a nucleotide between 170 and 907 bp, while ORF1C-ORF2R yielded a
product corresponding to the prototype in one of the remaining strains (E. faecalis F-KM6), suggesting a left-end Tn1546
deletion as far as a nucleotide between 907 and 1871. No
amplicons were obtained from the remaining eight isolates,
arguing for an orf1 deletion until a nucleotide between 1871
and 3174.
Tn1546 elements were also analyzed for the presence of the
G-to-T mutation at position 8234 in the vanX gene using
INV2-VANX1 and DdeI digestion of amplicons. Restriction
analysis revealed the vanX mutation in five E. faecium isolates,
three human intestinal (HI-AN9, HI-AN18, and HI-MI34) and
two pork (F-KM8 and F-KM9) isolates (Fig. 4).
Fisher’s test failed to evidence any association between
Tn1546 type and a particular source of GRE or a particular
enterococcal species (P ⬎ 0.5).
Genetic detection and expression of virulence determinants.
The 105 vanA enterococcal isolates were screened for the
presence of AS genes, gelE, cylB, and esp and tested for clumping after growth in the presence of pheromone-containing supernatants of E. faecalis JH2-2 and E. faecalis OG1RF and for
gelatinase and hemolysin production.
The presence of AS genes was determined by PCR using
primers AGG and ASA373. To identify the specific AS gene,
AGG amplicons were subjected to EcoRI restriction analysis,
together with those obtained with E. faecalis OG1RF(pCF10,
prgB) and E. faecalis OG1RF(pAD1, asa1) (Fig. 5). All tested
E. faecalis isolates carried at least one AS gene. In particular,
HI-AN23, HC-N22, and F-PM25 carried prgB, whereas HCVI4 was shown to contain both prgB and asa1 genes, as well as
asa373. Four strains showed a restriction profile with an additional fragment, and the corresponding AS genes were indicated as prgBⴱ (HC-UD6, F-KM6, and F-KM7) and asa1ⴱ
(HC-R35). HC-AN21 contained both prgBⴱ and asa1.
A total of 21 vanA isolates exhibited a positive clumping
reaction. Growth in the presence of pheromones gave rise to
different levels of aggregation: some strains generated moderate to large aggregates, whereas others elicited a barely detectable effect (Table 6). Clumps were particularly evident in six
(five human and one food) E. faecalis isolates, all containing
one or more of the AS genes tested (HC-VI4 [prgB, asa1, and
asa373], HC-AN21 [prgBⴱ and asa1], HC-AN22 [prgB], HCUD6 [prgBⴱ], HI-AN23 [prgB], and F-PM25 [prgB]). Clumps
were less pronounced in the remaining 15 isolates (8 human, 5
animal, and 2 food; 12 E. faecium, 2 E. durans and 1 E. gallinarum), none of which contained any of the AS genes tested.
Downloaded from http://aem.asm.org/ on February 16, 2016 by guest
FIG. 2. Plasmid profile (A) and vanA hybridization (B) of animal (lines 1 to 9) and human (lines 10 to 19) isolates. Line 1, E. durans A-VI4;
line 2, E. faecium A-PD33; line 3, E. durans A-VR5; line 4, E. faecium A-PD6; line 5, E. faecium A-VR8; line 6, E. faecium A-PD37; line 7, E.
faecium A-VI9; line 8, E. faecium A-VI10; line 9, E. faecium A-VR12; line 10, E. faecium HI-MI29; line 11, E. faecium HI-MI30; line 12, E. faecium
HI-MI57; line 13, E. faecium HI-MI58; line 14, E. faecium HI-MI31; line 15, E. faecium HI-MI32; line 16, E. faecium HI-MI27; line 17, E. faecium
HI-MI60; line 18, E. faecium HI-MI34; line 19, E. faecium HI-MI28. M, molecular size marker (Marker II; Roche).
3314
BIAVASCO ET AL.
APPL. ENVIRON. MICROBIOL.
The clumping-negative phenotype correlated with the presence of an additional EcoRI site in E. faecalis HC-R35 (asa1ⴱ),
F-KM6 (prgBⴱ), and F-KM7 (prgBⴱ).
gelE was detected in 28 isolates, of which eight (five human and
three food) were also Gel producers (Table 6). All the gelEpositive Gel producers were E. faecalis isolates and carried at least
one AS gene (F-KM6, F-KM7, F-PM25, HC-VI4, HC-AN21,
HC-AN22, HC-UD6, and HC-R35). No Gel production was detected in the remaining gelE-positive strains (17 E. faecium [APD1, A-VC2, A-PD35, A-PD6, A-VI9, A-VI10, A-VR12,
F-KM9, F-PM12, F-PM15, HC-R38, HI-VI1, HI-AN19, HIMI54, HI-MI30, HI-MI57, and HI-MI32], 2 E. durans [F-KM5
and F-PM20], and 1 E. gallinarum [A-PG15]). The five esp-positive strains (Table 6) included four E. faecium isolates (HCAN64, HC-VI2, HC-UD5, and HI-VI1) and an E. gallinarum
isolate (F-KM1). cylB was not detected in any of the 105 enterococcal isolates, all of which were negative for -hemolysis.
Biofilm formation. When the 105 vanA isolates were tested in
vitro for biofilm formation on abiotic surfaces, 10 strains were
seen to have a strong or weak ability to produce biofilm (Table 6).
Seven of these strains were E. faecalis (four human and three
food), two E. faecium (one human and one animal), and one E.
durans (animal). None of the four E. gallinarum strains were able
to form biofilm. Interestingly, the seven positive E. faecalis strains
(five strong and two weak producers) were also positive for prgB
and/or asa1 and gelE and were negative for esp. These features
were independent of the source of isolation. In contrast, the two
E. faecium weak biofilm producers and the only E. durans isolate
(a strong biofilm producer) were negative for all tested virulence
traits (Table 6).
Overall, strains possessing suspected virulence genes were
more frequent among clinical isolates than in the other reservoirs (P ⬍ 0.05), whereas there was no significant difference in
their occurrence between human intestinal, animal, and food
isolates (P ⬎ 0.6). However, the occurrence of strains carrying
multiple virulence factors was peculiar to clinical and food
reservoirs only.
DISCUSSION
Since the 1997 European Union ban forbidding the use of
avoparcin in animal feeds, the prevalence of GRE has de-
Downloaded from http://aem.asm.org/ on February 16, 2016 by guest
FIG. 3. PFGE of S1-digested (A) and I-CeuI-digested (C) total DNA and corresponding vanA (B and E) and 16S rRNA gene (D) hybridization. Lane 1, E. faecium HI-MI28; lane 2, E. faecium HI-MI34; lane 3, E. faecium HI-MI60; lane 4, E. faecium HI-MI32; lane 5, E. faecium
HI-MI31; lane 6, E. faecalis HI-MI58; lane 7, E. faecium HI-MI57; lane 8, E. faecium HI-MI30; lane 9, E. faecium HI-MI25; lane 10, E. faecium
HI-MI30; lane 11, E. faecium HI-MI25; lane 12, E. faecium HI-MI31; lane 13, E. faecium HI-MI32; lane 14, E. faecium HI-MI60; lane 15, E.
gallinarum A-BE48; lane 16, E. gallinarum F-PM3. M, low range marker (BioLabs).
VOL. 73, 2007
TYPING AND VIRULENCE OF vanA ENTEROCOCCI
3315
creased among farm animals and in the community (15, 34,
54), even though a readily detectable persistence of GRE in
avoparcin-exposed farm environments has been reported (1,
33). By contrast, the incidence of GRE in hospitals has remained substantially unchanged in northern Europe and has
actually increased in southern European countries (http://www
.earss.rivm.nl). The role of different reservoirs in the spread of
glycopeptide resistance is thus still unclear.
In this study, we compared GRE isolates of different origins
and geographic locations. GRE were initially identified at the
species level, PFGE typed, and analyzed for their van genotypes. Results showed different species prevailing in the different reservoirs. As expected, E. faecium was the most prevalent
species irrespective of the source, whereas E. faecalis was recovered only from human (mostly clinical) and food samples.
E. durans and E. gallinarum were only isolated from animal and
food samples. All isolates were confirmed to be vanA positive
and vanB negative.
PFGE results showed a polyclonal distribution of vanA isolates in the different reservoirs; however, the presence of some
clones in different reservoirs was observed. In particular, 26 E.
faecium isolates belonged to type M49; they were first isolated
in northern Italy in 1997 and since 2002 in northern and central
Italy, thus showing both a temporal and a geographic spread. A
different subtype characterized the different hospitals or towns
of isolation. The same clone was isolated from both clinical and
intestinal human samples, suggesting an ability of intestinal
isolates to act as pathogens. However, no difference in virulence determinants was detected between clinical and intestinal clonally related E. faecium isolates. Clinical isolates could
act as opportunistic pathogens or could have acquired virulence traits still to be characterized. Notably, the M49 type and
subtypes belong to ST 78, which has already been described as
epidemic in Italy (6). Type M39 was recovered over a limited
period of time (2002 to 2003) and geographic area (central
Italy) from human and pig intestines, suggesting an ability to
colonize both species. It was shown to belong to ST 18, which
has never been found in either human epidemic or animal
GRE according to a previous Italian study (6). Interestingly,
ST 18 and ST 78 both belong to the clonal complex 17 (CC17),
the first globally dispersed nosocomial-adapted clonal lineage
of E. faecium (56). However, while type M49 (ST 78) was
isolated from intestinal samples of inpatients only, type M39
(ST 18) was isolated from intestinal samples of inpatients as
well as in the community and in pigs. Isolates belonging to
types M7, M29, M42, D1, D5, and G2 were also recovered
from samples of different origins, demonstrating an occasional
clonal spread between different reservoirs also for E. durans
and E. gallinarum. M29 and M42, as well as other PFGE types
isolated from poultry in different geographic areas, carried the
purK type 6 allele, whereas all human hospital isolates (except
type M11) carried purK type 1, according to data reported by
others (6, 33, 50). Overall, these data strongly suggest that
human colonization by food and animal GRE is possible but
Downloaded from http://aem.asm.org/ on February 16, 2016 by guest
FIG. 4. Schematic representation of the Tn1546 prototype (A) and 11 different Tn1546-like elements (A* to D1) detected in the 101 vanA
isolates carrying a single vanA element. Locations of primers, ClaI target sites, and the mutation at position 8234 are indicated. Left-side deletions
(␦, deletion size) are indicated by dotted lines, insertions by gray boxes. The origin (HC, HI, A, and F) and number of isolates carrying the Tn1546
type are reported on the right. The labels of the Tn1546-like elements of Palepou et al. (43) that may correspond to those characterized in the
present study are reported in parentheses: A, A* (A); B* (D); B1 (D/M); B2 (M); B3 (P); C, C1 (B/C); C2 (no correspondence); C3 (H-L); D (E);
and D1 (Q-S).
3316
BIAVASCO ET AL.
that vertical transmission between different reservoirs is infrequent. Although the colonization might be transient (49, 57),
the possible transfer of resistance genes during this period
could be crucial.
Results of Tn1546 location analyses suggested an association
among the vanA location, species, and origin of isolates. In the
vast majority of our strains (98%), vanA was located on plasmids of either ⬍150 kb (all the E. faecalis and E. durans
strains) or ⬎150 kb (intestinal E. faecium, both human—including M49 and M39—and animal). Notably, plasmids of
⬎150 kb have already been described in an E. faecium clone
widely disseminated among pigs (1). These results suggest that
high-molecular-weight E. faecium plasmids might be involved
in intestinal colonization of both humans and animals, thus
contributing to the persistence of resistant strains. Moreover,
large plasmids are likely to be conjugative, thus contributing to
the horizontal transfer processes. A chromosomal vanA location was demonstrated in two of the four E. gallinarum strains,
suggesting that in this species the chromosome is a more common location than in other enterococcal species, as also reported previously (23).
Tn1546 typing showed a Tn1546 element, indistinguishable
from the prototype, in about 80% of the strains tested. The
remaining Tn1546-like elements displayed insertions or leftend deletions. This finding agrees with other data from Italian
vanA strains from different sources (5). In contrast, Tn1546like elements different from the prototype seem to be more
common in other countries (8, 26, 32, 46, 57). The same
Tn1546 type was found in clonally unrelated poultry, swine,
and human strains (Fig. 4), while different Tn1546 types were
found in isolates belonging to the same clone (Table 6), suggesting that horizontal gene transfer may have played a significant role in the spread of glycopeptide-resistant strains. The
finding of the G-to-T mutation at position 8234 of Tn1546 in
pork and human isolates, suggesting a relationship between
human and food vanA elements, supports this hypothesis.
When evaluated for virulence determinants and their expression, about half of the GRE showed at least one virulence
trait, gelE and the pheromone response being the most frequent. Gelatinase production was found in all clinical and food
E. faecalis isolates. Since gelatinase production has been more
frequently described in clinical isolates than in those from
other sources (11, 13), these results point to a link between
clinical and food reservoirs, as suggested by previous reports
(9, 47). Silent gelE was detected in isolates from other species
(E. faecium [particularly human and animal feces], E. durans
[food], and also E. gallinarum [animal feces]). gelE has been
documented frequently in E. faecalis, rarely in E. faecium and
E. durans (20, 24), but never in E. gallinarum. Thus, the spread
of gelE from E. faecalis by horizontal gene transfer might be
involved in the evolution of different pathogenic enterococcal
species. Lack of expression in species other than E. faecalis
might be explained by low levels or downregulation of gene
expression, an inactive gene product, or experimental conditions. Growth in the presence of E. faecalis sex pheromones
gave rise to clumps in all species, although the level of aggregation was higher in E. faecalis, suggesting a species-specific
response. On the other hand, AS genes (prevalently prgB) were
detected only in E. faecalis, with some strains harboring more
than one gene (prgB, asa1, and asa373). AS genes were also
detected in a few clumping-negative isolates. The presence in
some strains of prgBⴱ or asa1ⴱ correlated with the clumpingnegative phenotype, suggesting an inactive gene product.
Although it is frequently carried by pheromone-response
plasmids (24, 27), cylB was never detected; esp was only detected in a few E. faecium (human) and E. gallinarum (food)
isolates, arguing against the association of these virulence traits
with glycopeptide resistance. Although the ability to form biofilm was uncommon, it is worth noting that it was mostly
present in vanA E. faecalis isolates harboring other virulence
genes.
Overall, virulence studies of our vanA enterococci revealed
different trends in the occurrence of virulence determinants
among human, food, and animal isolates. Their higher incidence detected among human and food compared with animal
isolates was associated with the presence of E. faecalis isolates
carrying multiple virulence factors in the former reservoirs.
A similar virulence profile observed among clinical and food
E. faecalis isolates and the absence of multivirulent animal
enterococci suggest that food could be more closely involved
than animals in the spread of virulent GRE in humans. Moreover, the different features observed in enterococcal strains
isolated from breeding animals and animal food raise questions about the source of food contamination by vanA enterococci. On the other hand, GRE polyclonality suggests that
horizontal transfer of the vanA cluster, rather than clonal
spread, is responsible for their emergence and dissemination.
Subsequently, the presence of vanA, combined with one or
Downloaded from http://aem.asm.org/ on February 16, 2016 by guest
FIG. 5. EcoRI restriction analysis of AGG amplicons of nine E.
faecalis isolates and of E. faecalis OG1RF(pAD1, asa1) and E. faecalis
OG1RF(pCF10, prgB) reference strains. Lane 1, HI-AN23; line 2,
HC-VI4 (also asa373 positive); line 3, HC-AN22; line 4, HC-UD6; line
5, HC-R35; line 6, HC-AN21; line 7, F-KM6; line 8, F-KM7; line 9,
F-PM25; line 10, OG1RF(pAD1; asa1); and line 11, OG1RF(pCF10;
prgB). *, additional EcoRI site. M, GeneRuler 100-bp DNA Ladder
Plus marker (M-Medical Genenco).
APPL. ENVIRON. MICROBIOL.
VOL. 73, 2007
TYPING AND VIRULENCE OF vanA ENTEROCOCCI
3317
TABLE 6. PFGE and Tn1546 type, vanA location, genotype or phenotype of virulence, and slime production of 48 vanA enterococcal isolates
of different origins harboring one or more virulence traitsa
Origin (n) and strain
Species
Genotype or phenotype of virulence
PFGE
type
vanA
location
Tn1546
type
gelE
Gel
esp
AS genes
Clumping activity
Slime
production
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
faecium
faecium
faecium
faecium
faecium
faecium
faecium
faecium
faecium
faecium
faecium
faecium
faecalis
M39a
M49f
M49
M48
M36
M49e
M49f
M49m
M49a
M29
M44
M27
S5
P*
P* ⫹ Chr
P*
P
P
ND
P* ⫹ Chr
P*
P*
P
P
P
P
A
A/C2
A
C
B3
A
A
A
A
A
A
A
A
⫹
⫹
⫹
⫹
⫺
⫺
⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
prgB
⫺
⫺
⫺
⫺
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫹
⫹⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
WP
⫺
⫺
⫺
SP
Animal (15)
A-VE44
A-VI2
A-PD1
A-PD6
A-PD35
A-VI19
A-VI10
A-VR12
A-N19
A-N20
A-VR8
A-AN21
A-AN14
A-PD41
A-PG15
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
faecium
faecium
faecium
faecium
faecium
faecium
faecium
faecium
faecium
faecium
faecium
durans
durans
durans
gallinarum
M41
M22b
M25
M14
M38
M22a
M18
M32
M37
M35
M8
D1a
D2
D6
G1
P*
P
P
P
P
P
P
P
P
P
P
P
P
P
P
B3
A
A
A
A
A
A
A
A
B3
A
A
A
A
A
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫹
⫹
⫺
⫹
⫹
⫺
WP
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
SP
⫺
⫺
⫺
Food (11)
F-KM9
F-PM12
F-PM15
F-PM13
F-KM6
F-KM7
F-PM25
F-KM5
F-PM20
F-PM3
F-KM1
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
faecium
faecium
faecium
faecium
faecalis
faecalis
faecalis
durans
durans
gallinarum
gallinarum
M10
M45
M3a
M45a
S3
S3a
S7
D1c
D7
G1
G2
P
P
P
P
P
P
P
P
P
Chr
P
A*
A
A
A
B2
A
A
C2
B3
D1
A
⫹
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫺
prgB*
prgB*
prgB
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫹⫹⫹
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫺
SP
WP
SP
⫺
⫺
⫺
⫺
Human clinical (9)
HC-AN64
HC-UD5
HC-VI2
HC-R38
HC-VI4
HC-AN21
HC-AN22
HC-UD6
HC-R35
E.
E.
E.
E.
E.
E.
E.
E.
E.
faecium
faecium
faecium
faecium
faecalis
faecalis
faecalis
faecalis
faecalis
M24
M49b
M48
M49n
S6
S4
S8
S1
S2
P
P
P
P*
P
P
P
P
P
D
A
C
A
A
A
A
A
A
⫺
⫺
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫺
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
prgB asa1 asa373
prgB* asa1
prgB
prgB*
asa1*
⫺
⫺
⫹
⫺
⫹⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫺
⫺
⫺
⫺
⫺
⫺
SP
WP
⫺
SP
a
Abbreviations: P, ⬍150-kb plasmid; P*, ⬎150-kb plasmid; Chr, chromosome; Gel, gelatinase production; WP, weak producer (0.120 ⬍ optical density ⬍ 0.240);
SP, strong producer (optical density ⬎ 0.240); ND, not detected.
more virulence genes in the same genome, could have favored
particular clusters of E. faecium, e.g., type M49 in the hospital
environment.
The E. faecium isolates were generally devoid of virulence
determinants, albeit with notable exceptions. Although all E.
faecium strains lacked the AGG genes, several strains formed
clumps after pheromone induction. The same profile was occasionally observed in E. durans and E. gallinarum isolates.
This phenomenon might depend on the presence of AS/pheromone systems in these species, different from those of E.
Downloaded from http://aem.asm.org/ on February 16, 2016 by guest
Human intestinal (13)
HI-AN19
HI-MI32
HI-MI57
HI-VI1
HI-AN8
HI-AN15
HI-MI30
HI-MI54
HI-MI58
HI-N42
HI-N43
HI-N44
HI-AN23
3318
BIAVASCO ET AL.
ACKNOWLEDGMENTS
We are grateful to Gary Dunny for supplying E. faecalis strains
OG1RF and OG1RF(pCF10) and to Pier Sandro Cocconcelli for
OG1RF(pAD1). Thanks also to Annalisa Cavallero (Ospedale S. Raffaele, Milano, Italy), Esther Manso (Azienda Ospedaliera Umberto I,
Ancona, Italy), Giuseppina Scagnelli (Department of Infectious Diseases, San Bortolo Hospital, Vicenza, Italy), and Roberta di Rosa
(Department of Clinical Medicine, University La Sapienza, Roma,
Italy) for providing human GRE; to Annalisa Pantosti (Istituto Superiore di Sanità, Roma, Italy) and Francesca Clementi (Polytechnic
University of Marche, Ancona, Italy) for providing animal and food
strains; and to Roberta Fontana (University of Verona, Italy) and
Anna Grossato (University of Padova, Italy) for providing animal
strains. We are also grateful to Luigi Ferrante for his assistance with
statistical analyses and to Manuela Vecchi for sequencing experiments.
This study was supported by the European research project “Antimicrobial Resistance Transfer from and between Gram-Positive Bacteria of the Digestive Tract and Consequences for Virulence”
(ARTRADI), contract QLK2-CT-2002-00843.
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