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Antimicrobial Agents and Chemotherapy, September 1999, p. 2161-2164, Vol. 43, No. 9
Unité des Agents Antibactériens,
Institut Pasteur, 75724 Paris Cedex 15, France1;
Department of Biochemistry, University of Cambridge,
Cambridge CB2 1QW, United Kingdom2; and
MRL Pharmaceutical Services, Herndon, Virginia
201713
Received 25 March 1999/Returned for modification 2 June
1999/Accepted 21 June 1999
Enterococcus faecalis BM4405 was resistant to low
levels of vancomycin (MIC, 16 µg/ml) and was susceptible to
teicoplanin (MIC, 0.5 µg/ml). No PCR product was obtained when the
total DNA of this clinical isolate was used as a template with primers
specific for glycopeptide resistance genes vanA,
vanB, vanC, and vanD. However, a
604-bp PCR fragment was obtained when V1 and V2 degenerate primers were
used and total DNA was digested with HindIII as a template.
The product was cloned and sequenced. The deduced amino acid sequence
had greater identity (55%) with VanC than with VanA (45%), VanB
(43%), or VanD (44%). This was consistent with the fact that BM4405
synthesized peptidoglycan precursors that terminated in
D-serine residues. After induction with vancomycin, weak
D,D-dipeptidase and penicillin-insensitive
D,D-carboxypeptidase activities were detected
in cytoplasmic extracts of BM4405, whereas a serine racemase activity
was found in the membrane preparation. This new type of acquired
glycopeptide resistance was named VanE.
Glycopeptide antibiotics are used
for the treatment of infections caused by gram-positive bacteria. They
form a complex with the C-terminal
D-alanyl-D-alanine
(D-Ala-D-Ala) of peptidoglycan precursors and
block their incorporation into the bacterial cell wall (15).
Glycopeptide-resistant enterococci have a broad geographical
distribution and are phenotypically and genotypically heterogeneous. Three types of acquired resistance to glycopeptides have been described
in enterococci (3). VanA-type strains display high-level inducible resistance to both vancomycin and teicoplanin following acquisition of transposon Tn1546 or closely related elements
(2). VanB-type strains have variable levels of inducible
resistance to vancomycin only (3). VanD-type strains are
resistant to various levels of vancomycin and teicoplanin
(14). In strains of all three phenotypes, glycopeptide
resistance is due to synthesis of peptidoglycan precursors that
terminate in D-lactate (D-Lac) instead of
D-Ala (5).
The VanA, VanB, and VanD ligases synthesize the depsipeptide
D-Ala-D-Lac. In VanA- and VanB-type strains
three other enzymes are required for resistance: the VanH
dehydrogenase reduces pyruvate to D-Lac (6),
whereas the VanX D,D-dipeptidase and the
penicillin G-insensitive VanY
D,D-carboxypeptidase (3) prevent
synthesis of
UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala
(pentapeptide[Ala]). The VanA and VanB types of resistance, but not
the VanD type, are generally transferable to other enterococci by conjugation.
VanC-type resistance, characterized by low-level resistance to
vancomycin, is specific to Enterococcus gallinarum,
Enterococcus casseliflavus, and Enterococcus
flavescens (11, 13). The vanC gene product
synthesizes D-Ala-D-serine
(D-Ala-D-Ser), which is substituted for
D-Ala-D-Ala in peptidoglycan precursors
(18). Insertional inactivation of vanC in
E. gallinarum BM4174 led to vancomycin susceptibility and
exclusive synthesis of precursors that end in
acyl-D-Ala-D-Ala, indicating that intrinsically
resistant enterococci also produce a
D-Ala:D-Ala ligase and that vanC is necessary for expression of vancomycin resistance (7, 18). The level of synthesis of pentapeptide[Ala] in BM4174 is low due to
the presence of weak D,D-dipeptidase and
D,D-carboxypeptidase activities
(18). It has been shown recently (i) that production in
E. gallinarum BM4174 of VanXYc, a protein that
has both D,D-dipeptidase and
D,D-carboxypeptidase activities, allows
hydrolysis of the dipeptide D-Ala-D-Ala and
removal of the ultimate D-Ala from pentapeptide[Ala] and
(ii) that the presence of vanC1 and
vanXYc is sufficient for resistance when
D-Ser is added to the culture medium of Enterococcus faecalis JH2-2 into which the two genes had been introduced
(17). A membrane-bound serine racemase, VanT, which produces
D-Ser peptidoglycan synthesis in BM4174 has also been
characterized (1).
We present in this report evidence that E. faecalis BM4405,
which is resistant to low levels of vancomycin and which is susceptible to teicoplanin, is of a new glycopeptide resistance type which has
similarities with the intrinsic VanC type of resistance.
Strains.
E. faecalis BM4405 was isolated from the
peritoneal dialysis fluid of a patient in a Chicago, Ill., hospital who
was diagnosed with peritonitis and who had received vancomycin.
E. faecalis JH2-2 is a derivative of strain JH-2, which is
resistant to fusidic acid and rifampin (10).
Antibiotic susceptibility testing.
MICs were determined by
the agar dilution method on Mueller-Hinton agar (Diagnostics Pasteur,
Marnes-la-Coquette, France) with an inoculum of 104 CFU per
spot. Plates were incubated overnight at 37°C.
Filter mating.
Transfer of vancomycin resistance from BM4405
to the recipient strain E. faecalis JH2-2 was attempted by
filter mating (6) with selection on brain heart infusion
(BHI) containing rifampin (20 µg/ml), fusidic acid (10 µg/ml), and
spectinomycin (60 µg/ml) or vancomycin (4 µg/ml).
DNA amplification, cloning, and sequencing.
Identification
of strain BM4405 to the species level was performed by PCR
(8). A PCR assay with primers specific for resistance genes
vanA, vanB, vanC1, vanC2,
and vanD (8, 14) was used to determine the
glycopeptide resistance genotype of E. faecalis BM4405.
Amplifications were carried out in a final volume of 100 µl
containing 40 pmol of each oligonucleotide primer, 50 nmol of each
2'-5'-triphosphate deoxynucleoside, 100 ng of template DNA, and 2 U of
Taq DNA polymerase. The reactions were performed in a DNA
thermal cycler (Perkin-Elmer Cetus, Norwalk, Conn.) as described
previously (8, 14). Amplification of fragments internal to
genes encoding related ligases with degenerate V1 and V2 primers was
performed as described previously (7). Prior to
amplification, total DNA from BM4405 was digested for 2 h at 37°C with HindIII (Pharmacia LKB,
Saint-Quentin-en-Yvelines, France). PCR fragments were purified on
Microspin S-400 HR Columns (Pharmacia LKB), cloned into pCR2.1
(Invitrogen, Leek, The Netherlands), and sequenced by the
dideoxynucleotide chain termination method (19) with T7 DNA
polymerase (Pharmacia LKB) and Sequence analysis.
The programs included in the GCG package
(Genetics Computer Group, Madison, Wis.) were used for sequence
editing, translation, and alignment.
Analysis of peptidoglycan precursors.
Analysis of the
peptidoglycan precursors of BM4405 was performed as described
previously (16). The strain was grown in BHI with or without
vancomycin (8 µg/ml) to the midexponential phase (A600 = 1). Ramoplanin was added, and
incubation was continued for 20 min. The bacteria were harvested by
centrifugation (12,000 × g, 2 min, 4°C), and the
cytoplasmic precursors which had accumulated were extracted with 8%
trichloroacetic acid (15 min, 4°C), desalted, and analyzed by
high-performance liquid chromatography.
D,D-Dipeptidase and
D,D-carboxypeptidase activities.
The
enzymatic activities were assayed in BM4405 extracts as described
previously (4). The D-Ala released from
D-Ala-D-Ala by
D,D-peptidase and from pentapeptide terminating
in D-Ala (pentapeptide[Ala]) by
D,D-carboxypeptidase was determined by using
D-amino acid oxidase (12). Bacteria were lysed
by treatment with lysozyme (2 mg/ml) at 37°C, followed by sonication,
and the membrane fraction was pelleted (100,000 × g,
45 min). Activities were measured in the supernatant and in the
resuspended pellet fraction.
Serine racemase activity.
The assay of serine racemase was
carried out as described previously (1). Cell fractions were
prepared by osmotic lysis of bacteria treated with lysozyme (400 µg
ml Nucleotide sequence accession number.
The sequence was
submitted to GenBank and was assigned accession no. AF136925.
Characterization of strain BM4405.
Clinical isolate E. faecalis BM4405 was resistant to low levels of vancomycin (MIC, 16 µg/ml) but was susceptible to teicoplanin (MIC, 0.5 µg/ml); it was
also resistant to spectinomycin (MIC, 64 µg/ml). To identify BM4405
to the species level, primers specific for genes encoding
D-Ala:D-Ala ligases in enterococci were used (8), and an amplification product was obtained with the
primer pair specific for the E. faecalis ddl gene. To
determine the glycopeptide resistance genotype of BM4405 we used
primers specific for resistance genes vanA, vanB,
vanC1, vanC2, and vanD (8,
14), but no PCR product was obtained. Furthermore, in Southern
blots with nonstringent washing conditions, none of the probes specific
for these resistance genes hybridized with total DNA from BM4405. These
results indicate that BM4405 is an E. faecalis strain with a
newly acquired glycopeptide resistance genotype.
Determination of the glycopeptide resistance genotype of
BM4405.
The degenerate primers V1 and V2, which allow
amplification of fragments internal to genes that encode related
ligases (7), were used in a PCR with total DNA of BM4405 as
the template. A ca. 600-bp fragment was obtained, cloned into
Escherichia coli, and sequenced. The 10 clones analyzed
exhibited a sequence identical to that of part of the E. faecalis
ddl ligase gene. E. faecalis ddl has a
HindIII restriction site which is absent from the
vanA, vanB, vanC, and vanD
ligase resistance genes. After digestion of total BM4405 DNA with
HindIII, a PCR with V1 and V2 primers was performed and
a ca. 600-bp fragment was obtained with low efficiency. This PCR
product was digested with HindIII, to eliminate putative
partial digests of total DNA, and a second PCR was performed under the
same conditions. The reaction resulted, with high efficiency, in the
production of a ca. 600-bp fragment which was cloned, and both strands
were sequenced (Fig. 1). The deduced
amino acid sequence was compared with those of the host
D-Ala:D-Ala ligase, the
D-Ala:D-Lac ligases of VanA, VanB, and VanD
strains, and the VanC1 D-Ala:D-Ser ligase (Fig.
2); the percentages of identity were
calculated from this alignment (Table 1).
The sequence obtained had a higher degree of identity with the
corresponding portion of VanC1 (55%) than with that of VanA, VanB, or
VanD (from 43 to 45%). The motifs conserved in the related ligases
were present, and of the four amino acids that are invariably present
in the VanC-type D-Ala:D-Ser ligases (EKYQ, at
positions 142 to 145 [9]; Fig. 2 numbering), three
(EKY) were conserved. The 604-bp product used as a probe in a Southern
blot under stringent binding and washing conditions hybridized with
total DNA from BM4405 but not with total DNA from E. faecium
BM4147 (VanA), E. faecalis V583 (VanB), E. gallinarum BM4174 (VanC), or E. faecium BM4339 (VanD)
(data not shown). These results confirm that the 604-bp fragment is
internal to a new glycopeptide resistance gene that we have called
vanE. Primers specific for the new gene were synthesized and
used in an attempt to amplify fragments internal to genes encoding
related ligases from Enterococcus strains BM4147 (VanA),
V583 (VanB), BM4174 (VanC), BM4339 (VanD), and BM4405. A PCR fragment
of the expected size of 513 bp was obtained only with BM4405 DNA, and
direct sequencing of this product confirmed that the oligonucleotides
were specific for vanE. The 513-bp fragment hybridized only
with BM4405 DNA (data not shown).
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
VanE, a New Type of Acquired Glycopeptide
Resistance in Enterococcus faecalis BM4405
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
-35S-dATP (Amersham
Radiochemical Center). Specific amplification of a 513-bp fragment
internal to vanE was obtained with primers VANE1
(5'-TGTGGTATCGGAGCTGCAG-3') and VANE2
(5'-GTCGATTCTCGCTAATCC-3') under the following conditions:
30 s at 94°C, 30 s at 52°C, and 30 s at 72°C (30 cycles).
1) and M1 muramidase (70 µg ml1). The
membrane fraction was separated from the cytoplasm by centrifugation at
100,000 × g for 45 min.
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
View larger version (32K):
[in a new window]
FIG. 1.
Sequence of the 604-bp PCR product internal to the
vanE gene of E. faecalis BM4405 and the
corresponding amino acid sequence.
View larger version (60K):
[in a new window]
FIG. 2.
Alignment of the deduced partial amino acid sequence of
VanE and of the corresponding regions of VanA, VanB, VanC, VanD, and
Efa, the D-Ala:D-Ala ligase of E. faecalis. The amino acids conserved in all the sequences are
printed in boldface. Dots indicate gaps introduced to optimize sequence
similarity. The conserved motif EKY(Q/N) present in VanC-type and VanE
D-Ala:D-Ser ligases is underlined. GenBank
accession numbers are as follows: X56895 for VanA, U35369 for VanB,
M75132 for VanC, AF130997 for VanD, AF136925 for VanE, and U00457 for
the D-Ala:D-Ala ligase for E. faecalis.
TABLE 1.
Sequence identity between the deduced amino acid sequence
of VanE and related ligases
Transfer of vancomycin resistance. Attempts to transfer vancomycin resistance from BM4405 to E. faecalis JH2-2 by filter mating were unsuccessful. Only transfer of spectinomycin resistance, which is not associated with vancomycin resistance, was obtained.
Characterization of peptidoglycan precursors of BM4405.
To
analyze the cytoplasmic peptidoglycan precursors, cultures of E. faecalis BM4405, grown with or without vancomycin (8 µg/ml), were incubated in the presence of ramoplanin to inhibit cell wall synthesis after formation of the precursors. The results obtained indicated that, in the absence of vancomycin, UDP-MurNAc-pentapeptide was the unique precursor synthesized, whereas after incubation with
vancomycin, UDP-MurNAc-pentapeptide[Ser] and UDP-MurNAc-tetrapeptide were the main compounds produced (Table
2). These data indicate that resistance
in BM4405 is inducible by vancomycin and are consistent with the
finding that vanE is more closely related to vanC
than to other ligase genes (vanA, vanB, and
vanD). Incubation with vancomycin did not induce resistance
to teicoplanin.
|
D,D-Dipeptidase and
D,D-carboxypeptidase activities of E. faecalis BM4405.
D,D-Dipeptidase
(VanX) and D,D-carboxypeptidase (VanY)
hydrolyze the dipeptide D-Ala-D-Ala and remove
the terminal D-Ala residue of precursors ending in
acyl-D-Ala-D-Ala, respectively (3). After centrifugation at 100,000 × g the
D,D-dipeptidase activity in the supernatant of
lysed BM4405 that had been grown in the absence or in the presence (8 µg/ml) of vancomycin was assayed (Table 3). Cytoplasmic extracts from
vancomycin-induced E. faecalis BM4405 possessed weak
D,D-dipeptidase activity, comparable to that
found in E. gallinarum BM4174 (VanC) (16), and no
activity was detected in extracts from uninduced bacteria. No
D,D-carboxypeptidase activity was found in
membrane preparations of induced or uninduced cells, even in the
absence of penicillin G in the assay (Table 3). However, weak
D,D-carboxypeptidase activity was detected in
cytoplasmic extracts of induced BM4405 cells. This activity, which was
insensitive to penicillin G, could account for the presence of
tetrapeptide peptidoglycan precursors. Interestingly, the localization of D,D-carboxypeptidase activity in the
cytoplasm is similar to that in the VanC-type strain E. gallinarum BM4174 (18) but different from that of VanA-
and VanB-type strains, in which VanY is predominantly membrane bound.
It is possible that the D,D-peptidase and
D,D-carboxypeptidase activities of BM4405 are
encoded by a single gene, as in BM4174 (17).
|
Serine racemase activity. VanC-type resistance requires three proteins: VanC and VanXYc, which catalyze synthesis of D-Ala-D-Ser (18) and which eliminate precursors ending in D-Ala-D-Ala (17), and VanT, a membrane-bound serine racemase for production of D-Ser (1). BM4405 (VanE) synthesizes peptidoglycan precursors that end in D-Ala-D-Ser and therefore also requires a source of D-Ser. Serine racemase activity was present in the membrane fraction of BM4405 (Table 3), and the enzyme was inducible by vancomycin. The serine racemase activity was ca. 10-fold greater than that of BM4174, which could account for the higher ratio of pentapeptide[Ser]:tetrapeptide (9:1) present in cytoplasmic extracts of ramoplanin-inhibited BM4405 than in BM4174, for which the ratio was between 0.5:1 and 1:1 (data not shown). All the serine racemase activity was membrane bound, whereas alanine racemase activity was present almost exclusively in the cytoplasm (data not shown). The distribution of the alanine and serine racemases between the cytoplasm and the membrane fractions was identical in BM4405 (VanE) and BM4174 (VanC).
In conclusion, VanE-type glycopeptide resistance in E. faecalis BM4405 is due to synthesis of late peptidoglycan precursors ending in D-Ala-D-Ser. The VanC and VanE types of resistance are biochemically and phenotypically similar. The vanE gene cluster in BM4405 is under study.| |
ACKNOWLEDGMENT |
|---|
This work was supported in part by a Bristol-Myers Squibb Unrestricted Biomedical Research Grant in Infectious Diseases.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Unité des Agents Antibactériens, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France. Phone: (33) (1) 45 68 83 18. Fax: (33) (1) 45 68 83 19. E-mail: brunoper{at}pasteur.fr.
Present address: Laboratoire de Microbiologie, CHU de la Côte
de Nacre, 14033 Caen Cedex, France.
| |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
|
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0066-4804/99/$04.00+0
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Arias, C. A., Pena, J., Panesso, D., Reynolds, P.
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Boyd, D. A., Cabral, T., Van Caeseele, P., Wylie, J., Mulvey, M. R.
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Ambur, O.-H., Reynolds, P. E., Arias, C. A.
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Kawalec, M., Gniadkowski, M., Kedzierska, J., Skotnicki, A., Fiett, J., Hryniewicz, W.
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Lafaurie, M., Perichon, B., Lefort, A., Carbon, C., Courvalin, P., Fantin, B.
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Kawalec, M., Gniadkowski, M., Zaleska, M., Ozorowski, T., Konopka, L., Hryniewicz, W.
(2001). Outbreak of Vancomycin-Resistant Enterococcus faecium of the Phenotype VanB in a Hospital in Warsaw, Poland: Probable Transmission of the Resistance Determinants into an Endemic Vancomycin-Susceptible Strain. J. Clin. Microbiol.
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45: 986-989
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Lee, W. G., Jernigan, J. A., Rasheed, J. K., Anderson, G. J., Tenover, F. C.
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39: 1165-1168
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Dalla Costa, L. M., Reynolds, P. E., Souza, H. A. P. H. M., Souza, D. C., Palepou, M.-F. I., Woodford, N.
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44: 3444-3446
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McKessar, S. J., Berry, A. M., Bell, J. M., Turnidge, J. D., Paton, J. C.
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44: 3224-3228
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Goh, S. H., Facklam, R. R., Chang, M., Hill, J. E., Tyrrell, G. J., Burns, E. C. M., Chan, D., He, C., Rahim, T., Shaw, C., Hemmingsen, S. M.
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Kobayashi, K., Rao, M., Keis, S., Rainey, F. A., Smith, J. M. B., Cook, G. M.
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McGregor, K. F., Young, H.-K.
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44: 2341-2348
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Kawalec, M., Gniadkowski, M., Hryniewicz, W.
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Aarestrup, F. M.
(2000). Characterization of Glycopeptide-Resistant Enterococcus faecium (GRE) from Broilers and Pigs in Denmark: Genetic Evidence that Persistence of GRE in Pig Herds Is Associated with Coselection by Resistance to Macrolides. J. Clin. Microbiol.
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Okabe, T., Oana, K., Kawakami, Y., Yamaguchi, M., Takahashi, Y., Okimura, Y., Honda, T., Katsuyama, T.
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(2000). The Biopesticide Paenibacillus popilliae Has a Vancomycin Resistance Gene Cluster Homologous to the Enterococcal VanA Vancomycin Resistance Gene Cluster. Antimicrob. Agents Chemother.
44: 705-709
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