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Journal of Clinical Microbiology, June 2000, p. 2392-2394, Vol. 38, No. 6
Canadian Science Centre for Human and Animal
Health, Bureau of Microbiology, Laboratory Centre for Disease Control,
Health Canada, Winnipeg, Manitoba,1 and
Department of Microbiology, The Toronto Hospital,
University of Toronto, Toronto, Ontario,2 Canada
Received 16 August 1999/Returned for modification 24 January
2000/Accepted 15 March 2000
A single vanD-containing Enterococcus
faecium strain (N97-330) was isolated in Canada. The
vanD-containing region was cloned and sequenced.
Although the proteins have more than 96% identity to a previously
described vanD region in BM4339, the
vanSD gene contains a frameshift mutation that
leads to a predicted truncated protein. Furthermore, sequence analysis
of the ddl gene revealed the presence of an
IS982-like element (ISEfm1) which interrupted the D-Ala-D-Ala ligase. This suggested the
constitutive expression of the vanD operon, which was
confirmed. Pulsed-field gel electrophoresis fingerprinting demonstrated
that BM4339 was not related to N97-330 (>15 band differences).
Both strains contained multiple copies of the IS982-like element.
Vancomycin-resistant enterococci
produce modified precursors that terminate in either
D-alanyl-D-lactate
(D-Ala-D-Lac) or D-alanyl-D-serine
(D-Ala-D-Ser), which have a much lower affinity for glycopeptides than do unmodified precursors (2, 5). The
genetic basis for resistance lies in genes whose products have homology
to the bacterial D-Ala-D-Ala ligases, encoded
by ddl genes, which produce the dipeptide target for
glycopeptide antibiotics. High-level vancomycin resistance is conferred
either by the transferable, inducible VanA or VanB
D-Ala-D-Lac ligases (4, 11, 18) or
by the nontransferable, constitutive VanD D-Ala-D-Lac ligase, which thus far has been
described only for Enterococcus faecium BM4339 (7,
17) and E. faecium A902 (15). This report
describes the genetic characterization of vancomycin resistance in a
strain of E. faecium which is the first VanD-type strain
isolated in Canada.
A vancomycin-resistant enterococcus was isolated from a stool specimen
from a 59-year-old Ontario man who had had an orthoptic liver
transplant 46 days before and had received multiple courses of
antibiotics. Multiple attempts to amplify vanA,
vanB, and vanC1 using PCR with the previously
reported primer sets (8) were negative. Initial confirmation
of the isolate as a possible VanD-type strain was done courtesy of P. Courvalin (Institut Pasteur) using primers which amplify a 0.46-kb
vanD fragment (17). The strain (N97-330) was
identified as an E. faecium strain by using standard biochemical tests for strain identification (9). The MICs
(micrograms per milliliter) of a number of antibiotics as determined by
agar dilution (14) are shown in Table
1 and compared to those for the
vanD strains BM4339 and A902 (15, 17). Expression
of the vancomycin-resistance phenotype was not inducible in N97-330
(data not shown), suggesting constitutive expression of resistance
genes similar to that in BM4339 (17) but unlike that in A902
(15). Macrorestriction analysis was carried out by
separating ApaI- or SmaI-digested genomic DNA
by pulsed-field gel electrophoresis using 1.1% agarose gels and a
CHEF-DRIII (Bio-Rad, Hercules, Calif.) with the following pulse times:
1 to 10 s for 12 h followed by 1 to 35 s for 31 h
at 200 V in 0.5× Tris-borate-EDTA at 14°C. A comparison of the DNA
fingerprints of N97-330 and BM4339 (courtesy of F. Tenover, Centers for
Disease Control and Prevention) did not reveal any genetic relationship
between the two strains (13, 23). E. faecium
A902, which differed from BM4339 (
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Molecular Characterization of the vanD
Gene Cluster and a Novel Insertion Element in a Vancomycin-Resistant
Enterococcus Isolated in Canada
ABSTRACT
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6 band differences) (15), also appears not to be related to N97-330 (data not
shown).
TABLE 1.
Antibiograms of vanD-containing E. faecium strains N97-330, BM4339 (17), and A902 (15)
By using standard methods (20) and the 0.46-kb
vanD PCR product as a probe, two overlapping clones were
isolated from an N97-330 genomic library constructed in 
EMBL3
(Promega, Madison, Wis.). These clones were subjected to partial
sequence analysis (Fig. 1). The sequence
of the 5' end of the vanRD gene was obtained by
sequencing a PCR product amplified from N97-330 genomic DNA using a
primer designed based on a sequence upstream of BM4339 vanRD (7) and a primer from a
previously sequenced region of the N97-330 vanRD
gene. The complete nucleotide sequence of the 6,793-bp vanD
region is shown in Fig. 1A. The BLAST programs (1) (http://www.ncbi.nlm.nih/BLAST) were used to identify putative products of detected open reading frames (ORFs). Six genes were found
to have the same genetic organization as that of the vanD operon of E. faecium BM4339 (Fig. 1) (7).
Comparisons of the amino acid products from the two strains revealed
that pairs of homologous genes had at least 96% identity (data not
shown). Further comparisons of the vanD sequence from
N97-330 revealed 2.1 and 3.1% base pair differences with the genes
from BM4339 and E. faecium A902 (15),
respectively. These differences are less than those exhibited by the
three designated vanB subtypes (3.6 to 5%) (10, 16) but the genes may still be considered vanD
variants.
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Beginning 161 bp downstream of the vanXD stop
codon is an ORF of 909 bp (orfR1) coding for a putative
product of 302 amino acids that shows low but significant homology to
several regulatory proteins. The highest homology was with the YobV
protein (313 residues) from Bacillus subtilis (accession no.
AF027868), with which it has 30% identity. We have detected a region
of dyad symmetry beginning 10 bp downstream of the
vanXD stop codon that could form a putative
hairpin structure with a 
G of 96 kJ/mol that may play a regulatory
role (Fig. 1B). Transcription studies are needed to determine if
orfR1 is part of the vanD operon. Fifty base
pairs of sequence downstream of the BM4339 vanXD
is available (7), and an alignment with the N97-330 sequence
(data not shown) reveals significant divergence, with the regions
sharing only 40% identity. This may reflect a different genetic
location of the vanD region in BM4339 compared to that in
N97-330 or may be due to interstrain sequence divergence.
Comparison of the vanSD genes from BM4339 and N97-330 revealed that the latter has a 1-bp deletion at nucleotide position 670 of the BM4339 gene sequence, which results in a frameshift leading to, presumably, a truncated nonfunctional protein of 233 amino acids. The vanA and vanB operons are activated in the presence of an inducer when the VanR protein becomes phosphorylated either by the cognate autophosphorylated VanS (VanS ~ P) or by an unknown kinase (3, 22). In N97-330, the absence of a functional VanSD protein may lead to a high steady-state level of VanR ~ P and thus to constitutive expression of the vancomycin resistance operon, as growth studies have shown. Vancomycin resistance is constitutive in BM4339, although it carries an intact vanSD gene (7, 17).
During initial characterization of N97-330, PCR was carried out with
primers specific for the ddl gene of E. faecium
(8). A product approximately 1 kb larger than the expected
size was obtained (data not shown). Sequence analysis of 1,341 bp from this region revealed that a 1,041-bp insertion sequence (IS), defined
by 22-bp perfect terminal inverted repeats, had been inserted in the
ddl gene (Fig. 2A). Two
overlapping ORFs were identified in the IS, and putative translation
products showed between 38 and 43% identity with parts of the putative
transposase protein (Tnp) from the IS982 family of insertion
elements (12). Further analysis of the two ORFs revealed
that a translational frameshift in the region from bases 748 to 754 (Fig. 2A) could lead to the translation of a fusion protein of 302 residues which can be aligned to the Tnps of IS982 family
members. Control of transposition by programmed translational
frameshifting is common in a number of bacterial tnp genes,
though it has not been found in the IS982 family
(12). Alternatively, a frameshift mutation may have occurred in the tnp gene after the IS was acquired by the genome,
resulting in a truncated inactive protein. We propose naming this
element ISEfm1 (12). In order to determine the
number of copies of ISEfm1 in various strains of
enterococci, we probed ClaI digests with a fragment of the
tnp gene generated by PCR (Fig. 2B). Multiple copies exist
in the E. faecium vanA and vanD strains, whereas a vancomycin-sensitive strain (ATCC 19434) appears to have a single copy. The probe did not hybridize to the type strain E. faecalis ATCC 29212. Several faint bands visible on the
autoradiograph in the E. faecium lanes may be due to a low
level of homology between the probe and the tnp genes of
other IS elements found in E. faecium. It will be
interesting to expand this work to include additional species of
Enterococcus to determine if ISEfm1 is specific to E. faecium.
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Enterococci with impaired Ddl activity that require the presence of a glycopeptide for growth have been reported (6, 19, 21, 24). Although N97-330 appears to have a nonfunctional Ddl due to insertion of an IS element, a glycopeptide is not required for growth due to a frameshift mutation in vanSD, which most likely leads to the constitutive expression of the van operon.
Nucleotide sequence accession numbers. The complete nucleotide sequence of the 6,793-bp vanD region shown in Fig. 1A has been deposited in the GenBank database under accession number AF175293. The sequence of the ddl region containing ISEfm1 (Fig. 2A) has been deposited in the GenBank database under the accession number AF138282.
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ACKNOWLEDGMENTS |
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We gratefully acknowledge P. Courvalin for initial identification of a vanD amplicon from E. faecium N97-330 and R. Easy, R. Bosey, C. Murphy, and R. Hizon for valuable technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Canadian Science Centre for Human and Animal Health, Bureau of Microbiology, Laboratory Centre for Disease Control, Health Canada, 1015 Arlington St., Winnipeg, Manitoba, Canada R3E 3R2. Phone: (204) 789-2133. Fax: (204) 789-2018. E-mail: michael_mulvey{at}hc-sc.gc.ca.
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REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. |
Altschul, S. F.,
T. L. Madden,
A. A. Schaffer,
J. Zhang,
Z. Zhang,
W. Miller, and D. J. Lipman.
1997.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res.
25:3389-3402 |
| 2. |
Arthur, M., and P. Courvalin.
1993.
Genetics and mechanisms of glycopeptide resistance in enterococci.
Antimicrob. Agents Chemother.
37:1563-1571 |
| 3. |
Arthur, M.,
F. Depardieu,
G. Gerbaud,
M. Galimand,
R. Leclercq, and P. Courvalin.
1997.
The VanS sensor negatively controls VanR-mediated transcriptional activation of glycopeptide resistance genes of Tn1546 and related elements in the absence of induction.
J. Bacteriol.
179:97-106 |
| 4. |
Arthur, M.,
C. Molinas,
F. Depardieu, and P. Courvalin.
1993.
Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147.
J. Bacteriol.
175:117-127 |
| 5. | Arthur, M., P. Reynolds, and P. Courvalin. 1996. Glycopeptide resistance in enterococci. Trends Microbiol. 4:401-407[CrossRef][Medline]. |
| 6. | Baptista, M., F. Depardieu, P. Reynolds, P. Courvalin, and M. Arthur. 1997. Mutations leading to increased levels of resistance to glycopeptide antibiotics in VanB-type enterococci. Mol. Microbiol. 25:93-105[CrossRef][Medline]. |
| 7. |
Casadewall, B., and P. Courvalin.
1999.
Characterization of the vanD glycopeptide resistance gene cluster from Enterococcus faecium BM4339.
J. Bacteriol.
181:3644-3648 |
| 8. | Dutka-Malen, S., S. Evers, and P. Courvalin. 1995. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J. Clin. Microbiol. 33:24-27[Abstract]. |
| 9. | Facklam, R. R., and D. F. Sahm. 1995. Enterococcus, p. 308-314. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed. ASM Press, Washington, D.C. |
| 10. |
Gold, H. S.,
S. Ünal,
E. Cerenado,
C. Thauvin-Eliopoulos,
G. M. Eliopoulos,
C. B. Wennersten, and R. C. Moellering, Jr.
1993.
A gene conferring resistance to vancomycin but not teicoplanin in isolates of Enterococcus faecalis and Enterococcus faecium demonstrates homology with vanB, vanA, and vanC genes of enterococci.
Antimicrob. Agents Chemother.
37:1604-1609 |
| 11. | Handwerger, S., and J. Skoble. 1995. Identification of chromosomal mobile element conferring high-level vancomycin resistance in Enterococcus faecium. Antimicrob. Agents Chemother. 39:2446-2453[Abstract]. |
| 12. |
Mahillon, J., and M. Chandler.
1998.
Insertion sequences.
Microbiol. Mol. Biol. Rev.
62:725-774 |
| 13. |
Morrison, D.,
N. Woodford,
S. P. Barrett,
P. Sisson, and B. D. Cookson.
1999.
DNA banding pattern polymorphism in vancomycin-resistant Enterococcus faecium and criteria for defining strains.
J. Clin. Microbiol.
37:1084-1091 |
| 14. | National Committee for Clinical Laboratory Standards. 1997. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. Approved standard M7-A4. National Committee for Clinical Laboratory Standards, Wayne, Pa. |
| 15. | Ostrowsky, B. E., N. C. Clark, C. Thauvin-Eliopoulos, L. Venkataraman, M. H. Samore, F. C. Tenover, G. M. Eliopoulos, R. C. Moellering, Jr., and H. S. Gold. 1999. A cluster of VanD vancomycin-resistant Enterococcus faecium: molecular characterization and clinical epidemiology. J. Infect. Dis. 180:1177-1185[CrossRef][Medline]. |
| 16. |
Patel, R.,
J. R. Uhl,
P. Kohner,
M. K. Hopkins,
J. M. Steckelberg,
B. Kline, and F. R. Cockerill, III.
1998.
DNA sequence variation within vanA, vanB, vanC-1, and vanC-2/3 genes of clinical Enterococcus isolates.
Antimicrob. Agents Chemother.
42:202-205 |
| 17. | Perichon, B., P. Reynolds, and P. Courvalin. 1997. VanD-type glycopeptide-resistant Enterococcus faecium BM4339. Antimicrob. Agents Chemother. 41:2016-2018[Abstract]. |
| 18. | Quintiliani, R., and P. Courvalin. 1996. Characterization of Tn1547, a composite transposon flanked by IS16 and IS256-like elements, that confers vancomycin resistance in Enterococcus faecalis BM4281. Gene 172:1-8[CrossRef][Medline]. |
| 19. | Rosato, A., J. Pierre, D. Billot-Klein, A. Buu-Hoi, and L. Gutmann. 1995. Inducible and constitutive expression of resistance to glycopeptides and vancomycin dependence in glycopeptide-resistant Enterococcus avium. Antimicrob. Agents Chemother. 39:830-833[Abstract]. |
| 20. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 21. | Sifaoui, F., and L. Gutmann. 1997. Vancomycin dependence in a VanA-producing Enterococcus avium strain with a nonsense mutation in the natural D-Ala-D-Ala ligase gene. Antimicrob. Agents Chemother. 41:1409[Medline]. |
| 22. |
Silva, J. C.,
A. Haldimann,
M. K. Prahalad,
C. T. Walsh, and B. L. Wanner.
1998.
In vivo characterization of the type A and B vancomycin-resistant enterococci (VRE) VanRS two-component systems in Escherichia coli: a nonpathogenic model for studying the VRE signal transduction pathways.
Proc. Natl. Acad. Sci. USA
95:11951-11956 |
| 23. | Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239[Medline]. |
| 24. |
Van Bambeke, F.,
M. Chauvel,
P. E. Reynolds,
H. S. Fraimow, and P. Courvalin.
1999.
Vancomycin-dependent Enterococcus faecalis clinical isolates and revertant mutants.
Antimicrob. Agents Chemother.
43:41-47 |
Journal of Clinical Microbiology, June 2000, p. 2392-2394, Vol. 38, No. 6
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