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Antimicrobial Agents and Chemotherapy, September 1998, p. 2215-2220, Vol. 42, No. 9
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Glycopeptide Antibiotic Resistance Genes in
Glycopeptide-Producing Organisms
C. G.
Marshall,1
I. A. D.
Lessard,2
I.-S.
Park,2 and
G. D.
Wright1,*
Department of Biochemistry, McMaster
University, Hamilton, Ontario, Canada L8N
3Z5,1 and
Department of Biological
Chemistry and Molecular Pharmacology, Harvard Medical School,
Boston, Massachusetts 021152
Received 12 December 1997/Returned for modification 10 February
1998/Accepted 15 June 1998
 |
ABSTRACT |
The mechanism of high-level resistance to vancomycin in enterococci
consists of the synthesis of peptidoglycan terminating in
D-alanyl-D-lactate instead of the usual
D-alanyl-D-alanine. This alternate cell wall
biosynthesis pathway is ensured by the collective actions of three
enzymes: VanH, VanA, and VanX. The origin of this resistance mechanism
is unknown. We have cloned three genes encoding homologs of VanH, VanA,
and VanX from two organisms which produce glycopeptide antibiotics: the
A47934 producer Streptomyces toyocaensis NRRL 15009 and the
vancomycin producer Amycolatopsis orientalis C329.2. The
predicted amino acid sequences are highly similar to those found in
VRE: 54 to 61% identity for VanH, 59 to 63% identity for VanA, and 61 to 64% identity for VanX. Furthermore, the orientations of the genes,
vanH, vanA, and vanX, are identical
to the orientations found in vancomycin-resistant enterococci. Southern
analysis of total DNA from other glycopeptide-producing organisms,
A. orientalis 18098 (chloro-eremomycin producer), A. orientalis subsp. lurida (ristocetin producer), and
Amycolatopsis coloradensis subsp. labeda
(teicoplanin and avoparcin producer), with a probe derived from the
vanH, vanA, and vanX cluster from A. orientalis C329.2 revealed cross-hybridizing DNA in all
strains. In addition, the vanH, vanA,
vanX cluster was amplified from all glycopeptide-producing organisms by PCR with degenerate primers complementary to conserved regions in VanH and VanX. Thus, this gene
sequence is common to all glycopeptide producers tested. These results
suggest that glycopeptide-producing organisms may have been the
source of resistance genes in vancomycin-resistant enterococci.
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INTRODUCTION |
The glycopeptide antibiotic
vancomycin and the structurally related antibiotic teicoplanin are
considered to be the last lines of defense against a variety of serious
infections caused by gram-positive organisms, such as enterococci,
methicillin-resistant Staphylococcus aureus, and
Clostridium difficile (11). The recent rapid
emergence and spread of vancomycin-resistant enterococci (VRE) has
therefore become a grave concern of both medical practitioners and
their patients (22, 27).
The molecular target of glycopeptide antibiotics is the
D-alanyl-D-alanine
(D-Ala-D-Ala) terminus of growing
peptidoglycan, the rigid polymer which protects bacterial cells
from osmotic lysis. By binding to this terminal dipeptide, glycopeptide
antibiotics interfere with proper cell wall formation, which results in
eventual cell death (6). Glycopeptide-resistant organisms
avoid such a fate by modifying the drug's peptide target,
specifically, by modifying it to the depsipeptide
D-alanyl-D-lactate
(D-Ala-D-Lac) (40). The biosynthetic
machinery required to effect this transformation in
vancomycin-resistant Enterococcus faecium is found on a
transposable element, Tn1546, which incorporates five genes
necessary and sufficient to confer high-level inducible glycopeptide
resistance (2). Two of these gene products, VanR and VanS,
are required for the vancomycin-induced resistance response and are
members of a two-component regulatory system directing the
transcription of vanH, vanA, and vanX.
These genes encode the three proteins which are necessary for
D-Ala-D-Lac synthesis and are thus essential
for glycopeptide resistance (1). VanA is the pivotal enzyme,
producing the ester D-Ala-D-Lac instead of the
usual D-Ala-D-Ala dipeptide for incorporation into peptidoglycan precursors (9). VanH is an 
-keto acid
reductase (D-lactate dehydrogenase [D-LDH])
that supplies VanA with substrate by converting pyruvate into
D-lactate (9), while VanX is a highly specific
D,D-dipeptidase which hydrolyzes the
D-Ala-D-Ala generated by the endogenous cell
wall biosynthetic pathway but does not recognize
D-Ala-D-Lac (34, 43). This elegant
system is the means by which VRE can overcome current antibiotic
regimens. A second variant of this mechanism and gene structure is
encountered and distributed on plasmids (42), conjugative
chromosomal elements (32), or transposons (e.g.,
Tn1547 [31]) with the
D-Ala-D-Lac ligase designated VanB (18,
33).
The origin of the vanH, vanA/B, and
vanX (vanHAX) resistance cassette has
remained obscure. Both the intrinsically vancomycin-resistant lactic
acid bacteria, such as organisms from the genera
Leuconostoc, Pediococcus, and
Lactobacillus, and the glycopeptide-producing organisms are
potential reservoirs. Alternatively, the genes may have arisen through
mutations in homologous genes within enterococci or other organisms.
The vancomycin-resistant lactic acid bacteria such as Leuconostoc
mesenteroides have both the requisite
D-Ala-D-Lac ligase (15, 29) and
D-LDH (12) enzymes, although the
D-Ala-D-Lac ligases differ in their primary
sequence from VanA/B, notably in the 
-loop region which is critical
to catalysis (16). We have recently cloned a gene encoding a
VanA/B homolog, designated DdlM, from the glycopeptide
antibiotic-producing organism Streptomyces toyocaensis
NRRL 15009 (23). The striking homology between VanA/B and DdlM (62% identity, >77% similarity) suggested that these enzymes evolved from a common ancestor. Similarly, we reported a
segment of the ligase gene (ddlN) cloned from the
vancomycin-producing organism Amycolatopsis orientalis
C329.2 which also encoded a clear VanA/B homolog (23).
Importantly, the -loop segment is conserved between VanA, VanB,
DdlM, and DdlN. Thus, these results suggested that the VanA/B enzymes
originated in glycopeptide-producing organisms but that the origins of
the VanH and VanX accessory proteins were unknown. We now report on the
cloning and sequencing of genes encoding VanH and VanX homologs from
the same glycopeptide producers as well as the presence of
vanHAX gene clusters in several other
glycopeptide-producing organisms. The strong homology of the
gene products and the arrangement of the cloned genes indicate a
common origin for the vancomycin resistance genes in VRE and antibiotic-producing organisms.
| |
MATERIALS AND METHODS |
Cloning of vanX from S. toyocaensis NRRL
15009.
Cloning of a vanX gene from S. toyocaensis NRRL 15009 (vanXst) was
achieved by the isolation of a specific gene probe by PCR amplification
and degenerate deoxyoligonucleotide primers p1 and p2 designed to
amplify an internal vanX fragment of approximately 360 bp
(Table 1). PCR amplification reactions
contained S. toyocaensis NRRL 15009 total DNA as template
(500 ng), 1 U of Taq polymerase, each deoxynucleoside
triphosphate (dNTP) at a concentration of 0.4 mM, 1 µM primers, and 2 mM MgCl2. The 360-bp product was cloned into the pGEM-T
plasmid vector (Promega), and both strands were sequenced. To isolate a
clone encompassing the entire vanXst gene, the
probe fragment was randomly labeled with 32P by using the
Klenow fragment (4). Labeling reactions contained 200 ng of
template, 1 µl of each PCR primer, 1× Klenow buffer (10 mM Tris-Cl
[pH 7.5], 5 mM MgCl2, 7.5 mM dithiothreitol), and 10 U of
the Klenow fragment in a final volume of 55 µl. This probe was used
to screen Southern hybridization blots of total S. toyocaensis NRRL 15009 DNA which had been digested to completion
with several restriction enzymes in separate experiments. Hybridizing
fragments (5.2 kb) of DNA from digestion with PstI were
cloned into pBluescript II KS+ (Stratagene), transformed into
Escherichia coli DH5, and confirmed to contain the
vanXst gene by colony hybridization. A 5.2-kb
fragment was found to contain the vanXst gene
and was sequenced in its entirety.
The
vanH gene was located immediately upstream of the
ddlM gene, whose sequence we have reported previously
(
23).
Cloning of van genes from A. orientalis
C329.2.
Cloning of the A. orientalis C329.2
vanHAX cluster was achieved essentially as described for the
vanX gene of S. toyocaensis NRRL 15009. A
van gene probe was generated from A. orientalis C329.2 genomic DNA by degenerate PCR with primers p2 and p3
complementary to conserved sequences in vanA and
vanX (Table 1). Each amplification reaction mixture
consisted of 500 ng of total DNA from A. orientalis C329.2,
1× Taq buffer, 5% dimethyl sulfoxide, 2 mM
MgCl2, each dNTP at a concentration of 0.4 mM, 1 U of
Taq polymerase, and 1 µM primers. The 1.3-kb product was
cloned into pGEM-T (Promega), and this clone was used as a template to
generate a 32P-radiolabeled oligonucleotide probe with the
Klenow fragment. This probe was used to screen Southern blots of
restriction endonuclease-digested A. orientalis C329.2
genomic DNA to identify a 3.5-kb BamHI product containing
van genes. This fragment was cloned into pGEM-7Z and was
isolated by standard methods.
DNA sequencing.
The nucleotide sequence of the S. toyocaensis NRRL 15009 gene cluster was determined with a series
of nested deletions constructed by ExoIII-S1 nuclease
digestion and through the synthesis of specific oligonucleotide
primers. The gene cluster for A. orientalis C329.2 was
sequenced by primer walking. DNA cycle sequencing was performed by B. Allore on an Applied Biosystems automated DNA sequencer at the MOBIX
central facility at McMaster University.
Analysis of gene clusters in glycopeptide producing
organisms.
Total DNA was isolated from glycopeptide-producing
organisms, and 10 to 20 µg was separated on a 1% TAE
(Tris-acetate-EDTA)-agarose gel after digestion to completion with
BamHI. The DNA was blotted onto nitrocellulose by standard
methods. A vanHAX cluster oligonucleotide probe radiolabeled
with 32P was generated as described above by using the
cloned 3.5-kb BamHI fragment from A. orientalis
C329.2 as template and primers p3 and p4 (Table 1). This probe was
hybridized to genomic DNA restriction products, and the blot was washed
under stringent conditions prior to exposure.
PCR amplification of vanHAX from
glycopeptide-producing organisms.
The presence of the
vanHAX cluster in glycopeptide producers was also assessed
by amplification of the cluster by PCR. Degenerate oligonucleotide
primers complementary to conserved regions in VanH (primer p5) and VanX
(primer p2) were designed to amplify nearly the entire cluster (Table
1). The PCR mixtures (100 µl) contained 500 ng of total DNA, 1 U of
Taq polymerase, 1× Taq buffer, 5% dimethyl sulfoxide, 4 mM
MgCl2, each dNTP at a concentration of 0.4 mM, and 1 µM
primers. Forty cycles of 94, 55, and 72°C (1, 1, and 2 min,
respectively) were required to produce a visible band of approximately
2.6 kb. This product was isolated, applied to a 1% TAE agarose gel,
and blotted onto nitrocellulose. A 32P-labeled
vanHAX probe was generated from A. orientalis
C329.2 total DNA and primers p3 and p4 (Table 1).
Analysis of sequence data.
Contiguous sequences were
constructed by using the MegAlign software from DNA Star (Madison,
Wis.). Open reading frames (ORFs) were screened in all six frames by
using the program FRAME (8) to identify probable
streptomycete ORFs. Protein sequence alignments were performed by using
CLUSTAL W software (39), version 1.7, with the Blosum matrix
with a gap penalty of 10 and a gap extension of 0.05.
Nucleotide sequence accession numbers.
The nucleotide
sequences of vanHst and ddlM
(accession no. U82965), vanXst (accession no.
AF039028), and the gene cluster from A. orientalis
C329.2 (accession no. AF060799) have been deposited in GenBank.
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RESULTS AND DISCUSSION |
Cloning of van genes from S. toyocaensis
NRRL 15009 and A. orientalis C329.2.
S.
toyocaensis NRRL 15009 and A. orientalis C329.2
are gram-positive microbes which produce the "aglyco" glycopeptide
antibiotic A47934, a structural homolog of teicoplanin (44),
and vancomycin, respectively. We cloned and sequenced an expanse of
S. toyocaensis NRRL 15009 DNA spanning 2,650 bp and a
3,470-bp fragment of DNA from A. orientalis C329.2.
Each of these contains three ORFs whose products bear striking homology
to VanH, VanA (23), and VanX of VRE (Fig.
1). These genes have been designated
vanHst, ddlM (23), and
vanXst, respectively, in S. toyocaensis and vanHaov, ddlN,
and vanHaov, respectively, in A. orientalis C329.2. (This rather ponderous terminology is
necessitated by the fact that various A. orientalis
strains produce different glycopeptide antibiotics; thus, the subscript
ao designates A. orientalis and the
subscript v indicates that it is vancomycin producer.)
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FIG. 1.
Comparison of glycopeptide resistance gene clusters from
VRE and glycopeptide antibiotic-producing organisms.
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No genes encoding the homologs of the two-component regulatory elements
(VanR and VanS) of VRE, the
D,
D-carboxypeptidase (VanY),
or the
vancomycin-resistant enterococcus-associated proteins of
unknown
function (VanW or VanZ) were found in the DNA flanking
the
vanHAX cluster in either organism, although we did not
explicitly
screen for these and they may be located elsewhere in the
chromosome.
The 5' region immediately upstream of
vanHst contains an ORF which
encodes a putative
D-Ala-
D-Ala-adding enzyme (MurF) homolog,
which
we have designated MurX (
23), and part of a gene
homologous
to
femA which is required for the synthesis of
the penta-Gly bridge
that joins the peptidoglycan peptide strands and
is associated
with high-level methicillin resistance in staphylococci
(
13).
A 1.6-kb ORF, designated ORF1, is located 195 bp downstream from
vanXst. This ORF has associated promoter
30
and
10 sequences
and thus does not appear to be part of a
vanHst-ddM-vanXst polycistronic
message. The ORF1 amino acid sequence shows a high degree of homology
[P(N)10
170 to 10
42 by BLAST search] to
other bacterial proteins of unknown function.
Analysis against the
PROSITE database did not reveal any distinguishing
or revealing
features. Further sequencing of 2 kb of DNA downstream
of ORF1 did not
reveal any genes encoding proteins which could
obviously be ascribed to
peptidoglycan or glycopeptide antibiotic
biosynthesis.
No complete ORFs were detected in the regions flanking the
vanHAX cluster from
A. orientalis, although
only approximately
800 bp of flanking DNA was sequenced.
Putative streptomycete
20 (CGGGC) and
10 (CACATA) promoter
sequences (
36) are located upstream of
vanHst. No additional
promoter sequences were
found upstream of
ddlM or
vanXst;
thus,
these three genes appear to be driven by a single promoter, as
is
the case with the sequences of VRE. Putative
20 (CGGGG) and
10
(CCCATA) promoter sequences were also identified in the
A. orientalis C329.2 cluster. Additionally, a possible terminator
sequence located between bases 3228 and 3273 was identified.
The
vanHst gene terminates 10 bases within the
predicted 5' end of the
ddlM gene, and similarly,
vanHaov also terminates 10
bases within
ddlN (Fig.
2). These
observations directly parallel
the arrangement of
vanHA and
vanA in Tn
1546,
where the
vanHA termination
codon begins 5 bases
into the
vanA gene (
3) and in the VanB
gene
cluster where the
vanHB termination codon
similarly begins
5 bases into the
vanB gene (Fig.
2)
(
17).
Sequence analysis of vanH and vanX from
glycopeptide producing organisms.
The predicted amino acid
sequence of VanHst and VanHaov clearly
identify them as D-LDHs. Amino acid residues which are
thought to participate in catalysis based on site-directed mutagenesis studies (21, 37, 38) of D-LDHs, as well as the
three-dimensional structure of the Lactobacillus pentosus
D-LDH (35), are conserved in VanHst
and VanHaov. For example, a conserved Arg (Arg237 in VanHst and Arg255 in VanHaov) is predicted to
bind to the carboxylate and polarize the ketone carbonyl for reduction,
and an invariant His (His298 in VanHst and His316 in
VanHaov) is predicted to act as an active-site general acid
donating a proton during pyruvate reduction. Protein sequence alignment
of VanHst and VanHaov with the VanH enzymes
from VRE yielded overall amino acid identities of 51 to 55%, while
alignment with other D-LDHs resulted in identities ranging
from 26 to 31%. Phylogenetic analysis clearly places all the VanH
enzymes in a distinct branch of the D-LDH family (Fig. 3), an observation which parallels the
clustering of DdlM and DdlN with VanA and VanB within the
D-Ala-D-Ala ligase family (Fig. 4) (23).
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FIG. 3.
Phylogenetic relationship among VanH homologs as
calculated by the CLUSTAL W method (39). The tree was
generated with the PHYLIP program Drawtree (19).
Lact., Lactobacillus.
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FIG. 4.
Phylogenetic relationship among
D-Ala-D-Ala ligase homologs as calculated by
the CLUSTAL W method (39). The tree was generated with the
PHYLIP program Drawtree (19). The four major branches of the
tree are labeled A, B, C, and D.
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Phylogenetic analysis of the
D-Ala-
D-Ala
ligases indicates four major groups (Fig.
4): the A and B groups, which
are bona
fide
D-Ala-
D-Ala ligases but which
also contain the
D-Ala-
D-Lac
ligases from
lactic acid bacteria; the VanC group, which are
D-Ala-
D-Ser
ligases and which confer
constitutive low-level vancomycin resistance
in several organisms such
as
Enterococcus gallinarum and
Enterococcus casseliflavus (
14,
28); and the VanA group, which
consists
of the
D-Ala-
D-Lac ligases from VRE
and glycopeptide-producing
organisms.
Amino acid sequence alignment of VanX
st and
VanX
aov with the homologs from VRE confirms the presence of
the Zn
2+ binding ligands His116, Asp123, and His184 as well
as the putative
active site base Glu181 (
25) (Fig.
5). The VanX
st and
VanX
aov proteins show 61 to 63% identity (>75%
similarity) to the homologous
proteins in VRE.
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FIG. 5.
Overlap of the Zn2+ binding regions of VanX
proteins. The alignment was as calculated by the CLUSTAL W method
(39). The Zn2+ ligands are boxed, and the
putative active site base is indicated with an arrow.
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Identification of vanHAX gene clusters in other
glycopeptide antibiotic-producing organisms.
Several other
glycopeptide-producing organisms were screened for the presence of the
vanHAX genes by two methods. First, a part of the gene
cluster from A. orientalis C329.2 was amplified and
used to probe total DNA that had been isolated from several glycopeptide producers and digested to completion with
BamHI. A single hybridizing band was observed in
producers of chloro-eremomycin, ristocetin, vancomycin, and
teicoplanin-avoparcin (Fig. 6A),
indicating that these organisms harbor similar genes. Second, a PCR
screen was developed with degenerate primers complementary to conserved sequences in VanH and VanX. The presence of a vanHAX gene
cluster should give rise to an amplified product of approximately 2.7 kb. By using total DNA from several glycopeptide-producing organisms, a
band of the expected size was observed in all organisms tested, and the
sequence represented by this band was confirmed to encode the
vanHAX cluster by Southern hybridization with the
A. orientalis C329.2 van cluster probe (Fig.
6B). Thus, this gene cluster appears to be ubiquitously present in
glycopeptide-producing bacteria.
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FIG. 6.
The presence of the vanHAX gene cluster in
glycopeptide-producing organisms. (A) Southern blot of complete
BamHI digests of A. orientalis 18098 (chloro-eremomycin producer) (lane 1), A. orientalis
subsp. lurida (ristocetin producer) (lane 2), A. coloradensis subsp. labeda (teicoplanin and avoparcin
producer) (lane 3), and A. orientalis C329.2
(vancomycin producer) (lane 4). The blot was probed with a portion of
the ddlN gene amplified from A. orientalis
C329.2. (B) Results of PCR amplification of the vanHAX
cluster from glycopeptide-producing organism. The lane assignments and
the blot probe are the same as described for panel A. Numbers to
the left of the blots are in kilobases.
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Implications of the van gene cluster in
glycopeptide-producing organisms.
In addition to the considerable
similarity at the amino acid level noted above, the orientation and
alignment of the vanHst, ddlM, and
vanXst genes and the
vanHaov, ddlN, and
vanXaov genes in the glycopeptide producers is
identical to the orientation of the homologous genes in VRE (Fig. 1).
There are several implications of these findings. First, the fact that
glycopeptide producers possess this gene cluster implies that they use
a glycopeptide resistance mechanism which is similar to that found in
VRE. Second, the presence of the vanX
D,D-dipeptidase gene suggests that at some
point during growth, the organisms switch from producing the
conventional D-Ala-D-Ala peptidoglycan terminus
to producing D-Ala-D-Lac. This is supported by
our recent isolation of a dedicated D-Ala-D-Ala
ligase which is incapable of D-Ala-D-Lac
synthesis from S. toyocaensis NRRL 15009 cultures
that are not producing the A47934 glycopeptide antibiotic
(24).
More significant, however, are the evolutionary implications of these
results. Phylogenetic analysis reveals that the gene
products from
S. toyocaensis NRRL 15009,
A. orientalis C329.2,
and VRE form distinct subfamilies of
D-Ala-
D-Ala ligases and
D-LDHs.
Furthermore, the spatial arrangements of the genes are maintained
in
both the antibiotic-producing and clinically resistant organisms,
consistent with a common ancestry. Additionally, the signature
overlap
of the 5' end of the
vanA and homologous genes and the
3'
end of the
vanH genes is conserved. Thus, the cumulative
evidence
indicates that the origin of clinically relevant vancomycin
resistance
lies within the glycopeptide-producing organisms and not the
lactic
acid bacteria, which also possess
D-LDH and
D-Ala-
D-Lact ligase
enzymes but whose enzymes
are of a distinct lineage.
The G+C contents of the
S. toyocaensis NRRL 15009 and
A. orientalis C329.2 glycopeptide resistance genes are
65.3 and 63.6%,
respectively, while the genes of VRE are less purine
rich, with
44 and 49% purines for the VanA and VanB clusters,
respectively.
This weighs against recent direct transfer of the genes
from either
S. toyocaensis NRRL 15009 or
A. orientalis C329.2 to enterococci,
although other
glycopeptide-producing organisms, with different
G+C contents, may have
served as donor. We note that the G+C contents
of the VRE
vanH,
vanA/B, and
vanX elements are 5 to 10% higher
than those of the adjacent
vanR,
vanS,
vanY, and
vanZ (or
vanW)
genes, consistent with mobilization of the
vanH-vanA/B-vanX genes
as a unit from another source into a
context with appropriate
control elements. It is also interesting that
a difference in
G+C content is also found in the flanking DNA of the
gene clusters
cloned from the glycopeptide-producing organisms, but
here the
flanking sequences show higher G+C contents: 69.9% for
S. toyocaensis NRRL 15009 and 68.4% for
A. orientalis C329.2. Thus, the resistance
cluster may have been
acquired by these organisms as well. On
the basis of G+C content
analysis and in the absence of more sequence
information from
other glycopeptide antibiotic-producing organisms,
the
source of the
vanHAX cluster in VRE is not likely
to be an
actinomycete.
Alternatively, the resistance genes may have been acquired by
non-glycopeptide-producing soil organisms as a method of defense,
probably originally from a glycopeptide-producing organism, and
then
mobilized and passed on to other organisms, thus eventually
diluting
the original G+C content bias. It has been observed in
vanA-harboring
Oerskovia (G+C content of 70 to 75 mol%) that two
of the three nucleotide mutations present in
its version of
vanA serve to increase the G+C content
(
30). Whatever the mechanism
of mobilization of the
vanHAX cluster, if it originated in antibiotic-producing
organisms, the transfer was not a recent event. However, the use
of
vast quantities of the glycopeptide antibiotic avoparcin over
the past
two decades in European agriculture may have provided
the selective
pressure for this mobilization or the enrichment
of resistant organisms
in the environment. The fact that enterococci
harboring the
vanA gene cluster on transposons such as Tn
1546 can be isolated from farm environments in Europe (
5,
7)
but
not the United States (
10,
26) is telling. Furthermore,
the
observation that VRE first emerged in Europe may therefore
not be a
coincidence, although another reservoir for VRE in the
United States
cannot be ruled out (
26). For example, there has
been a
dramatic rise in the use of vancomycin in clinical settings
since the
early 1980s, especially in the United States (from 2,000
kg/year in
1984 to 11,200 kg/year in 1996 [
20]), and this
increase
in antibiotic use must be considered when discussing the rise
in the prevalence of VRE over the past decade. As an additional
cautionary note, it has recently been demonstrated that many antibiotic
formulations are contaminated with DNA from producing organisms
(
41); thus, a possible source for the dissemination of
resistance
genes exists, possibly as a contaminant in agricultural or
clinical
glycopeptide antibiotic formulations. The results described
here
should emphasize that caution must be employed in our use of
antibiotics
that, while not themselves clinically useful, have the
potential
to select for donors of resistance genes or preexisting
resistant
populations.
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ACKNOWLEDGMENTS |
This work was supported by an operating grant from the
Natural Sciences and Engineering Research Council of Canada (NSERC), by
a Medical Research Council of Canada Graduate (MRC) Studentship to
C.G.M., by an NSERC Postdoctoral Fellowship to I.A.D.L., and by an
MRC Scholar Award to G.D.W.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, McMaster University, 1200 Main St. W, Hamilton, ON,
Canada L8N 3Z5. Phone: (905) 525-9140, ext. 22943. Fax: (905) 522-9033. E-mail: wrightge{at}fhs.csu.mcmaster.ca.
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Abstract
Introduction
