Journal of Antimicrobial Chemotherapy (2008) 61, 838– 844
doi:10.1093/jac/dkn025
Advance Access publication 29 January 2008
Clinical implications of vancomycin-resistant Enterococcus faecium
(VRE) with VanD phenotype and vanA genotype
Jae-Hoon Song1,2*†, Kwan Soo Ko2,3†, Ji Yoeun Suh2†, Won Sup Oh1, Cheol-In Kang1,
Doo Ryeon Chung1, Kyong Ran Peck1, Nam Yong Lee4 and Wee Gyo Lee5
1
Division of Infectious Diseases, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul,
Korea; 2Asian-Pacific Research Foundation for Infectious Diseases (ARFID), Seoul, Korea; 3Department of
Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, Korea; 4Department of
Laboratory Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea;
5
Department of Laboratory Medicine, Ajou University School of Medicine, Suwon, Korea
Received 14 September 2007; returned 5 November 2007; revised 3 January 2008; accepted 4 January 2008
Objectives: To investigate the clinical implications of vancomycin-resistant Enterococcus faecium
(VRE) with VanD phenotype and vanA genotype (VanD-vanA VRE).
Methods: We tested in vitro and in vivo efficacies of teicoplanin against VanD-vanA VRE strains.
Change in teicoplanin MICs was monitored during incubation with teicoplanin. In vitro and in vivo
time –kill assay and survival analysis using a mouse peritonitis model were performed.
Results: Teicoplanin MICs of VanD-vanA VRE strains increased to 128 mg/L within 48 h when they were
cultured with 120 mg/L teicoplanin. In vitro and in vivo time –kill assay showed that VanD-vanA VRE
strains were not eliminated by 120 mg/L teicoplanin in contrast to vancomycin-susceptible E. faecium
and VanD-vanB strains. The survival rate of mice infected with VanD-vanA VRE strains treated with
teicoplanin was comparable with that of untreated mice.
Conclusion: Data suggest that teicoplanin would fail in the treatment of VanD type VRE infections if
the strains contained the vanA gene, which cannot be detected in the clinical microbiology laboratory.
Keywords: vancomycin-resistant enterococci, teicoplanin, VanD-vanA
Introduction
Enterococci have become a clinical concern with the emergence
of vancomycin-resistant Enterococcus faecium (VRE).1,2 The
importance of VRE has increased due to limited treatment
options and increased morbidity and mortality.3,4 VanA and
VanB are the most common phenotypes among six phenotypes
of glycopeptide resistance in enterococci.5 The VanA phenotype,
which is encoded by the vanA gene, is characterized by acquired
inducible and high-level resistance to vancomycin and teicoplanin. In contrast, the VanB phenotype is characterized by variable
levels of vancomycin resistance, and susceptibility to teicoplanin,
and is encoded by vanB.5,6
Recent studies reported VRE strains from Korea, Japan and
Taiwan with incongruence between phenotype and genotype,
which were susceptible to teicoplanin by in vitro test, despite
the presence of the vanA gene.7 – 12 It has been suggested
that point mutations in the sensor domain of the vanS gene in
Tn1546-like element (or vanA operon)7,8,10 or impairment of
accessory proteins VanY and VanZ9,11 would be the reason for
the loss of teicoplanin resistance in these strains. Our recent
report showed that 15% of VRE isolates from Korean hospitals
were teicoplanin-susceptible or -intermediate in spite of the
vanA gene,13 which is defined as VanD phenotype as in Naas
et al.14 Because these strains are reported to be susceptible or
intermediate to teicoplanin based on in vitro tests in the clinical
microbiology laboratory, clinicians would use teicoplanin for the
treatment of patients. However, clinical efficacy of teicoplanin
against these VanD-vanA VRE strains has not been investigated.
Given the increasing frequency of VanD-vanA VRE strains and
.....................................................................................................................................................................................................................................................................................................................................................................................................................................
*Corresponding author. Tel: þ82-2-3410-0320; Fax: þ82-2-3410-0328; E-mail: [email protected]
†J.-H. Song, K. S. Ko and J. Y. Suh contributed to this study equally.
.....................................................................................................................................................................................................................................................................................................................................................................................................................................
838
# The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
For Permissions, please e-mail: [email protected]
Clinical implications of VanD-vanA VREF
the very limited therapeutic options for VRE infections, this
would be an important issue in clinical practice. We report here
on the in vitro and in vivo evaluation of teicoplanin against VRE
strains with VanD phenotype and vanA genotype.
Materials and methods
Bacterial strains and antimicrobial susceptibility testing
E. faecium strains used in this study were isolated from tertiary care
hospitals in Korea (Samsung Medical Center, Kyungpook National
University Hospital and Chungnam National University Hospital)
(Table 1). Their genetic backgrounds were determined by multilocus
sequence typing (MLST), as described elsewhere.9,15 The genotype
for glycopeptide resistance was determined by the multiplex PCR
method.16 They were tested for in vitro vancomycin and teicoplanin
susceptibilities by evaluating MICs using the broth microdilution
method according to the CLSI.17 Six strains showed VanD phenotype (i.e. vancomycin-resistant and teicoplanin-susceptible or
-intermediate) with vanA gene. Although these six strains are classified as susceptible or intermediate to teicoplanin according to the
CLSI,17 teicoplanin MICs of 8 and 16 mg/L could be classified as
intermediate or even resistant, respectively, according to the
European Committee on Antimicrobial Susceptibility Testing.18 As
comparators, one strain with VanA phenotype and vanA genotype,
one strain with VanB phenotype and vanB genotype, and one
vancomycin-susceptible E. faecium (VSE) strain were tested.
Enterococcus faecalis ATCC 29212 and Staphylococcus aureus
ATCC 29213 strains were used as reference strains in vitro susceptibility testing.
Genetic analysis of Tn1546-like element
Genetic variations among van gene cluster (Tn1546-like element) of
VanD-vanA VRE strains were investigated by the PCR assay and
sequencing as in previous studies.11,19 Briefly, overlapping PCR
amplification of internal regions of Tn1546-like element was performed, and PCR fragments longer or shorter than that of the prototype vanA gene cluster of BM4147 were sequenced. The genetic
structures of Tn1546-like elements of the strains after exposure to
teicoplanin were also determined by PCR and sequencing of VanR,
VanS, VanX and VanY genes.
MIC changes after exposure to teicoplanin
Changes in teicoplanin MICs after exposure to teicoplanin were
determined for eight isolates of E. faecium (#18, #44, #88, 0-182,
06-12, 06-16, 07-16 and VSE-6800). Isolates were cultured in
Mueller –Hinton broth containing 120 mg/L teicoplanin and were
harvested after 2, 4, 6, 8, 12, 24, 48 and 72 h of culture. Teicoplanin
MIC for each isolate was determined using the broth microdilution
method according to the CLSI.17
In vitro time– kill assay
In vitro time–kill curves were determined for eight isolates of E.
faecium (#18, #44, #88, 01-182, 06-12, 06-16, 07-16 and
VSE-6800). Bacterial colonies were inoculated onto blood agar
plates and were incubated at 378C overnight. Each culture was suspended in saline solution (0.9% NaCl) and inoculated into freshly
prepared Mueller –Hinton broth, which contained 4 MIC, 8
MIC and 120 mg/L teicoplanin, adjusted to a final concentration of
106 cfu/mL. Viable counts were evaluated at 4, 8, 12, 24, 48 and
72 h with shaking incubation at 378C including antibiotic-free
control. All time–kill curve experiments were performed at least
three times and were averaged.
Establishment of mouse peritonitis model
To establish the mouse peritonitis model, 4- to 6-week-old female
outbred ICR mice with a mean weight of 20 g were purchased from
Orient Co. Ltd (Gyeonggi, Korea) and adapted to standardized
environmental conditions (temperature, 24 + 28C and humidity,
55 + 10%) for 1 week before the experiments. All animal procedures were performed in accordance with the institution’s guidelines for the humane handling, care and treatment of research
animals. Mice were administered intraperitoneally with 1 mL of an
overnight E. feacium culture suspended in saline solution (0.9%
NaCl) containing 5% pig mucin (Sigma, St Louis, MO, USA) to a
final cell density corresponding to the minimal lethal dose, which is
shown in Table 2. If untreated, mice died by 48–72 h. A 0.3 mL
Table 1. Characteristics of E. faecium strains used in this study
MIC (mg/L)
Strains
Origina
Phenotype/genotype
Source
vancomycin
teicoplanin
MLSTb
VSE-6800
#18
#88
#44
#28
01 –182c
06 –12c
06 –16c
07 –16c
SMC
SMC
SMC
SMC
SMC
SMC
KNUH
KNUH
CNUH
—
VanA/vanA
VanB/vanB
VanD/vanA
VanD/vanA
VanD/vanA
VanD/vanA
VanD/vanA
VanD/vanA
blood
blood
pus
blood
rectal swab
rectal swab
NA
NA
NA
0.5
.64
64
.64
.64
32
64
.64
.64
0.5
64
0.5
4
4
16
8
8
16
ST192 (15-1-1-1-1-7-1)
ST78 (15-1-1-1-1-1-1)
ST203 (15-1-1-1-1-20-1)
ST78 (15-1-1-1-1-1-1)
ST78 (15-1-1-1-1-1-1)
ST203 (15-1-1-1-1-20-1)
ST192 (15-1-1-1-1-7-1)
ST17 (1-1-1-1-1-1-1)
ST78 (15-1-1-1-1-1-1)
NA, not available.
a
SMC, Samsung Medical Center; KNUH, Kyungpook National University Hospital; CNUH, Chungnam National University Hospital.
b
Sequence type (ST, atpA-ddl-gdh-purK-gyd-pstS-adk).
c
These strains were analysed in a previous study.9
839
Song et al.
Table 2. Results of survival analysis
Survival rate (%)
Strain no.
Inoculum dose (log cfu/mL)
No. of mice tested
0h
12 h
24 h
36 h
48 h
60 h
72 h
Pa
VSE-6800
10.19
control (5)
treatment (10)
control (14)
treatment (15)
control (7)
treatment (10)
control (15)
treatment (14)
control (21)
treatment (20)
100
100
100
100
100
100
100
100
100
100
100
100
40.0
80.0
57.1
80.0
66.7
85.7
85.7
95.0
60.0
100
0
80.0
57.1
60.0
33.3
64.3
71.4
80.0
40.0
90.0
0
46.7
28.6
40.0
13.3
21.4
33.3
35.0
40.0
90.0
0
46.7
14.3
30.0
13.3
21.4
28.6
35.0
40.0
90.0
0
46.7
14.3
20.0
13.3
14.3
28.6
30.0
20.0
90.0
0
46.7
14.3
20.0
13.3
14.3
28.6
30.0
0.007
#88 (VanB –vanB)
9.70
#18 (VanA– vanA)
10.10
#44 (VanD– vanA)
10.20
#28 (VanD– vanA)
10.09
a
0.001
0.631
0.142
0.622
Kaplan–Meier method comparing between control and treatment groups.
dose of teicoplanin (40 mg/kg) was given subcutaneously to infected
mice 3 h after infection. Mice were observed for 72 h following
inoculation and the teicoplanin treatment. The concentration of teicoplanin was adjusted to 120 mg/L, which is the peak level of teicoplanin treatment in human serum based on previous experiments.20
When teicoplanin 40 mg/kg was injected into the mouse, the peak
level of teicoplanin (120 mg/L) in mouse serum was achieved; teicoplanin concentrations were determined microbiologically by an
agar-dilution method.
In vivo time– kill assay
The in vivo time–kill curves were determined by inoculating
various enterococcal strains into mice in each teicoplanin treatment
group. Mice were infected as in the mouse peritonitis model. Four
enterococcal strains were used: VSE-6800, #18 (VanA-vanA), #44
(VanD-vanA) and #88 (VanB-vanB). Teicoplanin (40 mg/kg) was
administered subcutaneously 3 h after infection to achieve the peak
concentration of 120 mg/L. Untreated mice were used as controls.
Blood samples were obtained from three infected mice by cardiac
puncture at 0, 6, 12, 24, 48 and 72 h after treatment with teicoplanin. Samples were plated on blood agar with appropriate dilution for
the determination of viable bacterial cell counts after overnight incubation in 5% CO2.
Survival analysis
Survival of mice was evaluated for five groups with different enterococcal strains: VSE-6800, #18 (VanA-vanA), #28 (VanD-vanA), #44
(VanD-vanA) and #88 (VanB-vanB). Five to 21 mice were prepared
as described earlier for each strain and controls. As for in vivo
time–kill growth curve determination, 40 mg/kg of teicoplanin was
administered subcutaneously 3 h after infection and re-administered
once a day. Untreated mice were used as controls. The cumulative
per cent survival (%S) was estimated at 12, 24, 36, 48, 60 and 72 h
after administration of teicoplanin. Evaluation of survival rate differences between the treatment group and the control group was performed using the Kaplan– Meier method. A P value of less than
0.05 was considered significant. SPSS for Windows (version 11.5
software package; SPSS Inc., Chicago, IL, USA) was used for the
analysis.
Results
Genetic analysis of Tn1546-like element of VRE strains
Genetic backgrounds of E. faecium isolates, which were determined using MLST, showed that they belonged to ST78 and its
single locus variants (Table 1). ST78 is a single locus variant of
ST17 and thus related to the internationally disseminated
lineage, CC-17.9 Structures of Tn1546-like elements, which
account for glycopeptide resistance of vanA genotype in
Enterococcus species, are shown in Figure 1. All isolates except
#28 and 06-12 showed different structures. This indicates that
loss of teicoplanin resistance in VanD-vanA VRE strains did not
occur by horizontal transfer of an impaired Tn1546-like
element, but by independent deletion of vanY or vanZ associated
with insertion of IS1216V. No point mutations in vanS were
found in any strain, but an IS1216V element was inserted within
vanS in strain 06-16.
MIC changes after exposure to teicoplanin
Teicoplanin MICs did not change significantly in enterococcal
strains with congruent phenotype and genotype. VSE and
VanB-vanB VRE strains retained susceptibility to teicoplanin
(MICs, both 1 mg/L) after exposure to 120 mg/L teicoplanin.
Exposure to teicoplanin resulted in only a 2-fold increase in
MIC, from 64 to 128 mg/L, in VanA-vanA VRE strain.
However, significant increases in teicoplanin MICs were
observed in VanD-vanA VRE strains after exposure to teicoplanin. Within 48 h of exposure to 120 mg/L teicoplanin, teicoplanin MICs increased from 4 –16 to 128 mg/L. This suggests that
VanD-vanA VRE strains could acquire high-level resistance to
teicoplanin, if they are exposed to teicoplanin, or that teicoplanin leads to the selection of the mutants. The acquired teicoplanin resistance was preserved after removal of the selective
pressure of teicoplanin.
In vitro time – kill analysis
The in vitro time –kill curve showed that the growth of VSE and
VanB-vanB VRE strains was inhibited by more than 2 logs by
840
Clinical implications of VanD-vanA VREF
Figure 1. Structures of Tn1546-like elements of six VanD-vanA VRE isolates used in this study. The location of genes and the direction of transcription of
normal Tn1546-like element of a strain BM4147 are shown at the top. Grey arrows represent IS elements. Dashed arrows are genes with interruption by IS
elements. The positions of IS insertion sites are shown at the corresponding sites.
120 mg/L teicoplanin, whereas VanD-vanA and VanA-vanA
VRE strains were not inhibited by this concentration of teicoplanin (Figure 2). Although a VanA-vanA strain was inhibited by
8 MIC of teicoplanin (512 mg/L), this concentration is more
than four times the achievable peak concentration (120 mg/L) in
humans.
In vivo time – kill analysis
The results of in vivo growth curve experiments were demonstrated in Figure 3. In vivo time –kill analysis showed that all
mice infected with E. faecium strains died within 48 h and bacterial cell numbers did not decrease if they were not treated with
teicoplanin. VSE and VanB-vanB VRE strains were eradicated
by teicoplanin after 48 h of treatment, and the infected mice survived. However, VanA-vanA and VanD-vanA VRE strains were
not eradicated from blood of the infected mice despite teicoplanin treatment. The mice infected with VanA-vanA or
VanD-vanA VRE strains used in the in vivo time – kill experiments died within 48 h irrespective of treatment with
teicoplanin.
Survival analysis
The Kaplan – Meier survival analysis showed that teicoplanin
treatment has significantly increased the survival rate of the
mice infected with VSE (P ¼ 0.007) or VanB-vanB VRE strains
(P ¼ 0.001). However, the survival rate of mice infected with
VanA-vanA or VanD-vanA VRE strains treated with 120 mg/L
teicoplanin was comparable with that of untreated controls
(Table 2).
Discussion
In clinical practice, only a few antimicrobial agents can be used
for the treatment of VRE infections, and these include quinupristin –dalfopristin, linezolid and teicoplanin. Teicoplanin could
be one of the therapeutic options for teicoplanin-susceptible
VRE infections either alone or in combination with clindamycin,
rifampicin and ampicillin.4,21,22 However, because it has been
also described that VanS(B) gene mutations mediate constitutive
or teicoplanin-inducible expression of resistance,23 combination
therapy with teicoplanin and aminoglycopeptides is more highly
recommended. In a surveillance study in Korean hospitals,
15.3% of VRE isolates showed teicoplanin susceptibility despite
vanA genotype.13 Most of them are thought to emerge sporadically to date due to the different structure of Tn1546-like
elements. However, it is possible that VanD-vanA VRE strains
disseminate clonally both between and within hospitals because
they belong to the same clonal complex, CC-78. A nosocomial
outbreak VRE showing susceptibility or intermediate resistance
to teicoplanin associated with vanA genotype was reported in a
French hospital.14
Because VanD-vanA VRE strains showed phenotypic susceptibility to teicoplanin by in vitro tests, teicoplanin could be
selected for use in the clinical practice. Data from this study,
however, suggested that teicoplanin therapy will fail if VanB- or
VanD-type VRE strains contain the vanA gene. According to
our in vitro time – kill assay, teicoplanin was not active against
VanD-vanA VRE strains. These strains also acquired high-level
resistance after exposure to teicoplanin without any structural
change in the Tn1546-like element, which was determined by
PCR and sequencing of VanR, VanS, VanX and VanY genes. In
a mouse peritonitis model, teicoplanin failed to eradicate
VanD-vanA VRE strains. These results are consistent with the
841
Song et al.
Figure 2. The results of in vitro time– kill analysis in E. faecium isolates. Open diamonds, 0 MIC; open squares, 4 MIC; open triangles, 8 MIC; open
circles, 120 mg/L.
previous studies using a rabbit endocarditis model, in which
VanD-type E. faecium with a vanD glycopeptide resistance
operon was not cured by teicoplanin and in vivo emergence of
teicoplanin-resistant mutants in VanB-type E. faecalis occurred
during teicoplanin treatment.24,25
VanD-vanA VRE strains may contain heterogeneous populations consisting of VanA-vanA and vancomycin-susceptible or
VanB-vanB cells. Recently, Naas et al.14 showed that the low
teicoplanin MIC for VRE strains with the vanA gene is due to
insertion of IS16 in the vanY gene, resulting in heterogeneous
expression of vancomycin resistance. Heterogeneous expression
of vancomycin resistance resulted from heterogeneous E.
faecium populations, which consisted of susceptible and highly
resistant cells. In our study, no heterogeneous colonies showing
teicoplanin and vancomycin resistances were detected, when
they were tested using teicoplanin and vancomycin Etest strips
(AB Biodisk, Solna, Sweden). In these strains, vanB gene could
not be amplified by multiplex PCR assay for the genotyping of
842
Clinical implications of VanD-vanA VREF
Figure 3. The results of in vivo growth curve experiments. Filled marks indicated mice groups treated with teicoplanin and empty marks indicated control
groups. In some strains, no values could be represented because all mice died.
glycopeptide resistance. In addition, genetic structures of
Tn1546-like elements determined from colonies, which survived
after exposure to teicoplanin, did not change. Thus, the
VanD-vanA VRE strains included in our study may not contain
heterogeneous populations.
Although the results in this study may be strain-dependent,
data from the current study suggest that phenotypic characteristics based on in vitro susceptibility tests in VRE isolates
cannot be the sole guide to the selection of antimicrobial agents
in clinical practice. Genotypic characteristics of glycopeptide
resistance in enterococci should also be considered in the treatment of VRE infection. Generally, however, genotyping of VRE
strains is not routinely performed in most clinical microbiology
laboratories. Given these in vitro and in vivo data about
VanD-vanA VRE strains, genotyping for glycopeptide resistance
would be required in the hospitals where VanD-vanA VRE
strains emerge.
In summary, we showed that teicoplanin was not effective
against VanD-vanA VRE strains both in vitro and in vivo,
despite its phenotypic susceptibility to teicoplanin. When teicoplanin is considered to treat severe VanD-type VRE infections,
genotyping of glycopeptide resistance is warranted.
Funding
This study was partly supported by the Korea Research
Foundation Grant funded by the Korean Government
(MOEHRD) (KRF-2005-015-E00195) and the Korean Food and
Drug Administration (KFDA).
Transparency declarations
None to declare.
References
1. Bonten MJM, Willems R, Weinstein RA. Vancomycin-resistant
enterococci: why are they here, and where do they come from? Lancet
Infect Dis 2001; 1: 314– 25.
2. Harbarth S, Cosgrove S, Carmeil Y. Effects of antibiotics on
nosocomial epidemiology of vancomycin-resistant enterococci.
Antimicrob Agents Chemother 2002; 46: 1619–28.
3. Lodise TP, McKinnon PS, Tam VH et al. Clinical outcomes for
patients
with
bacteremia
caused
by
vancomycin-resistant
Enterococcus in a level 1 trauma center. Clin Infect Dis 2002; 34:
922 –9.
4. Malathum K, Murray BE. Vancomycin-resistant enterococci:
recent advances in genetics, epidemiology and therapeutic options.
Drug Resist Updat 1999; 2: 224 –43.
5. Woodford N. Epidemiology of the genetic elements responsible
for acquired glycopeptide resistance in enterococci. Microb Drug Resist
2001; 7: 229 –34.
6. Cetinkaya Y, Falk P, Mayhal CG. Vancomycin-resistant enterococci. Clin Microbiol Rev 2000; 13: 686–707.
7. Eom JS, Hwang IS, Hwang BY et al. Emergence of vanA genotype vancomycin-resistant enterococci with low or moderate levels of
teicoplanin resistance in Korea. J Clin Microbiol 2004; 42: 1785–6.
8. Hashimoto Y, Tanimoto K, Ozawa Y et al. Amino acid substitutions in the VanS sensor of the VanA-type vancomycin-resistant
Enterococcus strains result in high-level vancomycin resistance and
low level teicoplanin resistance. FEMS Microbiol Lett 2000; 185:
247 –54.
9. Ko KS, Baek JY, Lee JY et al. Molecular characterization of
vancomycin-resistant Enterococcus faecium isolates from Korea. J Clin
Microbiol 2005; 43: 2303–6.
10. Lauderdale TL, McDonald LC, Shiau YR et al.
Vancomycin-resistant enterococci from humans and retail chickens in
Taiwan with unique VanB phenotype–vanA genotype incongruence.
Antimicrob Agents Chemother 2002; 46: 525–7.
11. Lee WG, Huh JY, Cho SR et al. Reduction in glycopeptide
resistance in vancomycin-resistant enterococci as a result of vanA
cluster rearrangements. Antimicrob Agents Chemother 2004; 48:
1379–81.
12. Oh WS, Ko KS, Song JH et al. High rate of resistance to quinupristin–dalfopristin in Enterococcus faecium clinical isolates from
Korea. Antimicrob Agents Chemother 2005; 49: 5176–8.
13. Song JH, Ko KS, Oh WS et al. High frequency of vancomycinresistant Enterococcus faecium isolates with VanB phenotype and
vanA genotype in Korean hospitals. Diagn Microbiol Infect Dis 2006;
56: 410–06.
14. Naas T, Fortineau N, Sanaoudj R et al. First nosocomial outbreak of vancomycin-resistant Enterococcus faecium expressing a
VanD-like phenotype associated with a vanA genotype. J Clin
Microbiol 2005; 43: 3642–9.
843
Song et al.
15. Homan WL, Tribe D, Poznanski S et al. Multilocus sequence
typing scheme for Enterococcus faecium. J Clin Microbiol 2002; 40:
1963–71.
16. Bell JM, Paton JC, Turnidge J. Emergence of vancomycinresistant enterococci in Australia: phenotype and genotype characteristics of isolates. J Clin Microbiol 1998; 36: 2187– 90.
17. Clinical and Laboratory Standards Institute. Performance
Standards for Antimicrobial Susceptibility Testing: Fifteenth
Informational Supplement M100-S16. CLSI, Wayne, PA, 2006.
18. European Committee on Antimicrobial Susceptibility Testing
(EUCAST). European Antimicrobial MIC Breakpoints Through
EUCAST and the National Breakpoint Committees. Basel: EUCAST,
2004. http://www.srga.org/eucastwt/bpsetting.htm (15 November 2007,
date last accessed).
19. Huh JY, Lee WG, Lee K et al. Distribution of insertion sequences
associated with Tn1546-like elements among Enterococcus faecium
isolates from patients in Korea. J Clin Microbiol 2004; 42: 1897–902.
20. Peetermans WE, Hoogeterp JJ, Hazekamp-van Dokkum AM
et al. Antistaphylococcal activities of teicoplanin and vancomycin in vitro
and in an experimental infection. Antimicrob Agents Chemother 1990;
34: 1869–74.
21. Linden PK. Treatment options for vancomycin-resistant enterococcal infections. Drugs 2002; 62: 425–41.
22. Losonsky GA, Wolf A, Schwalbe RS et al. Successful treatment
of meningitis due to multiply resistant Enterococcus faecium with a
combination of intrathecal teicoplanin and intravenous antimicrobial
agents. Clin Infect Dis 1994; 19: 163–5.
23. Baptista M, Depardieu F, Reynolds P et al. Mutations leading to
increased levels of resistance to glycopeptide antibiotics in VanB-type
enterococci. Mol Microbiol 1997; 25: 93–105.
24. Lefort A, Garry L, Depardieu F et al. Influence of VanD type
resistance on activities of glycopeptides in vitro and in experimental
endocarditis due to Enterococcus faecium. Antimicrob Agents
Chemother 2003; 47: 3515–8.
25. Lefort A, Arthur M, Depardieu F et al. Expression of
glycopeptide-resistance gene in response to vancomycin and teicoplanin in the cardiac vegetations of rabbits infected with VanB-type
Enterococcus faecalis. J Infect Dis 2004; 189: 90 – 7.
844