Skip to main content
PMC home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1998 Sep;42(9):2215–2220. doi: 10.1128/aac.42.9.2215

Glycopeptide Antibiotic Resistance Genes in Glycopeptide-Producing Organisms

C G Marshall 1, I A D Lessard 2, I-S Park 2, G D Wright 1,*
PMCID: PMC105782  PMID: 9736537

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.

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 α-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 ω-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 ω-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 DH5α, 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.

TABLE 1.

Sequences of PCR primers used in this study

Primera DNA sequence Associated protein Target amino acid consensus sequence
p1 (fwd) 5′-CA(T/C) TC(G/C) CGI GGI (T/A)C(C/G) (A/G)C(C/G) ATC GAC-3′ VanX HSRG(S/T)(A/T)ID
p2 (rev) 5′-G(T/A)A GTG CCA CCA (T/C)T(T/C)-3′ VanX EWWHY
p3 (fwd) 5′-GGI GAG GAC GGI (T/A)(C/G)I (C/A)TI CAG GG-3′ VanA GEDGAMQG
p4 (rev) 5′-GTG AAI CC(G/C) GGI A(T/G)I GTG TT-3′ VanA NT(L/I)PGFT
p5 (fwd) 5′-GGI TGC/T GGI T/A/CC/GG/C GAC GAG/A GC-3′ VanH GCG(S/P)DEA
Open in a new tab
a

fwd, forward; rev, reverse. 

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.

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.)

FIG. 1.

FIG. 1

Open in a new tab

Comparison of glycopeptide resistance gene clusters from VRE and glycopeptide antibiotic-producing organisms.

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 Tn1546, 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).

FIG. 2.

FIG. 2

Open in a new tab

Overlap of vanH and vanA homolog genes.

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).

FIG. 3.

FIG. 3

Open in a new tab

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.

FIG. 4.

FIG. 4

Open in a new tab

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.

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 VanXst and VanXaov with the homologs from VRE confirms the presence of the Zn2+ binding ligands His116, Asp123, and His184 as well as the putative active site base Glu181 (25) (Fig. 5). The VanXst and VanXaov proteins show 61 to 63% identity (>75% similarity) to the homologous proteins in VRE.

FIG. 5.

FIG. 5

Open in a new tab

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.

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.

FIG. 6.

FIG. 6

Open in a new tab

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.

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 Tn1546 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.

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.

REFERENCES

  • 1.Arthur M, Molinas C, Courvalin P. The VanS-VanR two-component regulatory system controls synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147. J Bacteriol. 1992;174:2582–2591. doi: 10.1128/jb.174.8.2582-2591.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Arthur M, Molinas C, Depardieu F, Courvalin P. Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147. J Bacteriol. 1993;175:117–127. doi: 10.1128/jb.175.1.117-127.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Arthur M, Molinas C, Dutka-Malen S, Courvalin P. Structural relationship between the vancomycin resistance protein VanH and 2-hydroxycarboxylic acid dehydrogenases. Gene. 1991;103:133–134. doi: 10.1016/0378-1119(91)90405-z. [DOI] [PubMed] [Google Scholar]
  • 4.Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K. Current protocols in molecular biology. New York, N.Y: John Wiley & Sons, Inc.; 1994. [Google Scholar]
  • 5.Bager F, Madsen M, Christensen J, Aarestrup F M. Avoparcin used as a growth promoter is associated with the occurrence of vancomycin-resistant Enterococcus faecium on Danish poultry and pig farms. Prev Vet Med. 1997;31:95–112. doi: 10.1016/s0167-5877(96)01119-1. [DOI] [PubMed] [Google Scholar]
  • 6.Barna J C J, Williams D H. The structure and mode of action of glycopeptide antibiotics. Annu Rev Microbiol. 1984;38:339–357. doi: 10.1146/annurev.mi.38.100184.002011. [DOI] [PubMed] [Google Scholar]
  • 7.Bates J, Jordens J Z, Griffiths D T. Farm animals as a putative reservoir for vancomycin-resistant enterococcal infection in man. J Antimicrob Chemother. 1994;34:507–516. doi: 10.1093/jac/34.4.507. [DOI] [PubMed] [Google Scholar]
  • 8.Bibb M J, Findlay P R, Johnson M W. The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences. Gene. 1984;30:157–166. doi: 10.1016/0378-1119(84)90116-1. [DOI] [PubMed] [Google Scholar]
  • 9.Bugg T D H, Wright G D, Dutka-Malen S, Arthur M, Courvalin P, Walsh C T. Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA. Biochemistry. 1991;30:10408–10415. doi: 10.1021/bi00107a007. [DOI] [PubMed] [Google Scholar]
  • 10.Coque T M, Tomayko J F, Ricke S C, Okhyusen P C, Murray B E. Vancomycin-resistant enterococci from nosocomial, community, and animal sources in the United States. Antimicrob Agents Chemother. 1996;40:2605–2609. doi: 10.1128/aac.40.11.2605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Cunha B A. Vancomycin. Med Clin N Am. 1995;79:817–831. doi: 10.1016/s0025-7125(16)30041-4. [DOI] [PubMed] [Google Scholar]
  • 12.Dartois V, Phalip V, Schmitt P, Divies C. Purification, properties and DNA sequence of the d-lactate dehydrogenase from Leuconostoc mesenteroides subsp. cremoris. Res Microbiol. 1995;146:291–302. doi: 10.1016/0923-2508(96)81052-7. [DOI] [PubMed] [Google Scholar]
  • 13.de Lencastre H, de Jonge B L, Matthews P R, Tomasz A. Molecular aspects of methicillin resistance in Staphylococcus aureus. J Antimicrob Chemother. 1994;33:7–24. doi: 10.1093/jac/33.1.7. [DOI] [PubMed] [Google Scholar]
  • 14.Dutka-Malen S, Molinas C, Arthur M, Courvalin P. Sequence of the vanC gene of Enterococcus gallinarum BM4174 encoding a d-alanine:d-alanine ligase-related protein necessary for vancomycin resistance. Gene. 1992;112:53–58. doi: 10.1016/0378-1119(92)90302-6. [DOI] [PubMed] [Google Scholar]
  • 15.Elisha B G, Courvalin P. Analysis of genes encoding d-alanine:d-alanine ligase-related enzymes in Leuconostoc mesenteroides and Lactobacillus spp. Gene. 1995;152:79–83. doi: 10.1016/0378-1119(94)00692-l. [DOI] [PubMed] [Google Scholar]
  • 16.Evers S, Casadewall B, Charles M, Dutka-Malen S, Galimand M, Courvalin P. Evolution of structure and substrate specificity in d-alanine:d-alanine ligases and related enzymes. J Mol Evol. 1996;42:706–712. doi: 10.1007/BF02338803. [DOI] [PubMed] [Google Scholar]
  • 17.Evers S, Courvalin P. Regulation of VanB-type vancomycin resistance gene expression by the VanSB-VanRB two-component regulatory system in Enterococcus faecalis V583. J Bacteriol. 1996;178:1302–1309. doi: 10.1128/jb.178.5.1302-1309.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Evers S, Sahm D F, Courvalin P. The vanB gene of vancomycin-resistant Enterococcus faecalis V583 is structurally related to genes encoding d-Ala:d-Ala ligases and glycopeptide-resistance proteins VanA and VanC. Gene. 1993;124:143–144. doi: 10.1016/0378-1119(93)90779-3. [DOI] [PubMed] [Google Scholar]
  • 19.Felsenstein J. PHYLIP (Phylogeny Inference Package), version 3.5c. 1993. Distributed by the author. Department of Genetics, University of Washington, Seattle. [Google Scholar]
  • 20.Kirst H A, Thompson D G, Nicas T I. Historical yearly usage of vancomycin. Antimicrob Agents Chemother. 1998;42:1303–1304. doi: 10.1128/aac.42.5.1303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Kochhar S, Chuard N, Hottinger H. Glutamate 264 modulates the pH dependence of the NAD(+)-dependent d-lactate dehydrogenase. J Biol Chem. 1992;267:20298–20301. [PubMed] [Google Scholar]
  • 22.Leclercq R, Courvalin P. Resistance to glycopeptides in enterococci. Clin Infect Dis. 1997;24:545–554. doi: 10.1093/clind/24.4.545. [DOI] [PubMed] [Google Scholar]
  • 23.Marshall C G, Broadhead G, Leskiw B, Wright G D. d-Ala-d-Ala ligases from glycopeptide antibiotic-producing organisms are highly homologous to the enterococcal vancomycin-resistance ligases VanA and VanB. Proc Natl Acad Sci USA. 1997;94:6480–6483. doi: 10.1073/pnas.94.12.6480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Marshall C G, Wright G D. The glycopeptide antibiotic producer Streptomyces toyocaensis NRRL 15009 has both d-alanyl-d-alanine and d-alanyl-d-lactate ligases. FEMS Microbiol Lett. 1997;157:295–299. doi: 10.1111/j.1574-6968.1997.tb12788.x. [DOI] [PubMed] [Google Scholar]
  • 25.McCafferty D G, Lessard I A, Walsh C T. Mutational analysis of potential zinc-binding residues in the active site of the enterococcal d-Ala-d-Ala dipeptidase VanX. Biochemistry. 1997;36:10498–10505. doi: 10.1021/bi970543u. [DOI] [PubMed] [Google Scholar]
  • 26.McDonald L C, Kuehnert M J, Tenover F C, Jarvis W R. Vancomycin-resistant enterococci outside the health-care setting: prevalence, sources, and public health implications. Emerg Infect Dis. 1997;3:311–317. doi: 10.3201/eid0303.970307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Murray E. Vancomycin-resistant enterococci. Am J Med. 1997;102:284–293. doi: 10.1016/S0002-9343(99)80270-8. [DOI] [PubMed] [Google Scholar]
  • 28.Park I S, Lin C H, Walsh C T. Bacterial resistance to vancomycin: overproduction, purification, and characterization of VanC2 from Enterococcus casseliflavus as a d-Ala-d-Ser ligase. Proc Natl Acad Sci USA. 1997;94:10040–10044. doi: 10.1073/pnas.94.19.10040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Park I S, Walsh C T. d-Alanyl-d-lactate and d-alanyl-d-alanine synthesis by d-alanyl-d-alanine ligase from vancomycin-resistant Leuconostoc mesenteroides. Effects of a phenylalanine 261 to tyrosine mutation. J Biol Chem. 1997;272:9210–9214. doi: 10.1074/jbc.272.14.9210. [DOI] [PubMed] [Google Scholar]
  • 30.Power E G M, Abdulla Y H, Talsania H G, Spice W, Aathithan S, French G L. VanA genes in vancomycin-resistant clinical isolates of Oerskovia turbata and Arcanobacterium (Cornebacterium) haemolyticum. J Antimicrob Chemother. 1995;36:595–606. doi: 10.1093/jac/36.4.595. [DOI] [PubMed] [Google Scholar]
  • 31.Quintiliani R J, Courvalin P. Characterization of Tn1547, a composite transposon flanked by the IS16 and IS256-like elements, that confers vancomycin resistance in Enterococcus faecalis BM428. Gene. 1996;172:1–8. doi: 10.1016/0378-1119(96)00110-2. [DOI] [PubMed] [Google Scholar]
  • 32.Quintiliani R J, Courvalin P. Conjugal transfer of the vancomycin resistance determinant vanB between enterococci involves the movement of large genetic elements from chromosome to chromosome. FEMS Microbiol Lett. 1994;119:359–363. doi: 10.1111/j.1574-6968.1994.tb06913.x. [DOI] [PubMed] [Google Scholar]
  • 33.Quintiliani R J, Evers S, Courvalin P. The vanB gene confers various levels of self-transferable resistance to vancomycin in enterococci. J Infect Dis. 1993;167:1220–1223. doi: 10.1093/infdis/167.5.1220. [DOI] [PubMed] [Google Scholar]
  • 34.Reynolds P E, Depardieu F, Dutka-Malen S, Arthur M, Courvalin P. Glycopeptide resistance mediated by enterococcal transposon Tn1546 requires production of VanX for hydrolysis of d-alanyl-d-alanine. Mol Microbiol. 1994;13:1065–1070. doi: 10.1111/j.1365-2958.1994.tb00497.x. [DOI] [PubMed] [Google Scholar]
  • 35.Stoll V S, Kimber M S, Pai E F. Insights into substrate binding by d-2-ketoacid dehydrogenases from the structure of Lactobacillus pentosusd-lactate dehydrogenase. Structure. 1996;4:437–447. doi: 10.1016/s0969-2126(96)00049-4. [DOI] [PubMed] [Google Scholar]
  • 36.Strohl W R. Compilation and analysis of DNA sequences associated with apparent streptomycete promoters. Nucleic Acids Res. 1992;20:961–974. doi: 10.1093/nar/20.5.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Taguchi H, Ohta T. Essential role of arginine 235 in the substrate-binding of Lactobacillus plantarumd-lactate dehydrogenase. J Biochem. 1994;115:930–936. doi: 10.1093/oxfordjournals.jbchem.a124441. [DOI] [PubMed] [Google Scholar]
  • 38.Taguchi H, Ohta T. Histidine 296 is essential for the catalysis in Lactobacillus plantarumd-lactate dehydrogenase. J Biol Chem. 1993;268:18030–18034. [PubMed] [Google Scholar]
  • 39.Thompson J D, Higgins D G, Gibson T J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Walsh C T, Fisher S L, Park L-S, Prahalad M, Wu Z. Bacterial resistance to vancomycin: five genes and one missing hydrogen bond tell the story. Chem Biol. 1996;3:21–28. doi: 10.1016/s1074-5521(96)90079-4. [DOI] [PubMed] [Google Scholar]
  • 41.Webb V, Davies J. Antibiotic preparations contain DNA: a source of drug resistance genes? Antimicrob Agents Chemother. 1993;37:2379–2384. doi: 10.1128/aac.37.11.2379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Woodford N, Jones B L, Baccus Z, Ludlam H A, Brown D F. Linkage of vancomycin and high-level gentamicin resistance genes on the same plasmid in a clinical isolate of Enterococcus faecalis. J Antimicrob Chemother. 1995;35:179–184. doi: 10.1093/jac/35.1.179. [DOI] [PubMed] [Google Scholar]
  • 43.Wu Z, Wright G D, Walsh C T. Overexpression, purification, and characterization of VanX, a d,d-dipeptidase which is essential for vancomycin resistance in Enterococcus faecium BM4147. Biochemistry. 1995;34:2455–2463. doi: 10.1021/bi00008a008. [DOI] [PubMed] [Google Scholar]
  • 44.Zmijewski M J, Jr, Fayerman J T. Glycopeptides. In: Vining L C, Stuttard C, editors. Genetics and biochemistry of antibiotic production. Boston, Mass: Butterworth-Heinemann; 1994. pp. 269–281. [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES