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ABSTRACT: Enterococcus faecium UCN71, isolated from a blood culture, was resistant to low levels of vancomycin (MIC, 16 μg/ml) but susceptible to teicoplanin (MIC, 0.5 μg/ml). No amplification was observed with primers specific for the previously described glycopeptide resistance ligase genes, but a PCR product corresponding to a gene called vanN was obtained using degenerate primers and was sequenced. The deduced VanN protein was related (65% identity) to the d-alanine:d-serine VanL ligase. The organization of the vanN gene cluster, determined using degenerate primers and by thermal asymmetric interlaced (TAIL)-PCR, was similar to that of the vanC operons. A single promoter upstream from the resistance operon was identified by rapid amplification of cDNA ends (RACE)-PCR. The presence of peptidoglycan precursors ending in d-serine and d,d-peptidase activities in the absence of vancomycin indicated constitutive expression of the resistance operon. VanN-type resistance was transferable by conjugation to E. faecium. This is the first report of transferable d-Ala-d-Ser-type resistance in E. faecium.
Antimicrobial Agents and Chemotherapy 08/2011; 55(10):4606-12. · 4.84 Impact Factor
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ABSTRACT: Inducible vancomycin resistance in enterococci is due to a sophisticated mechanism that combines synthesis of cell wall peptidoglycan precursors with low affinity for glycopeptides and elimination of the normal target precursors. Although this dual mechanism, which involves seven genes organized in two operons, is predicted to have a high fitness cost, resistant enterococci have disseminated worldwide. We have evaluated the biological cost of VanB-type resistance due to acquisition of conjugative transposon Tn1549 in Enterococcus faecium and Enterococcus faecalis. Because fitness was dependent on the integration site of Tn1549, an isogenic set of E. faecalis was constructed to determine the cost of inducible or constitutive expression of resistance or of carriage of Tn1549. A luciferase gene was inserted in the integrase gene of the transposon to allow differential quantification of the strains in cocultures and in the digestive tract of gnotobiotic mice. Both in vitro and in vivo, carriage of inactivated or inducible Tn1549 had no cost for the host in the absence of induction by vancomycin. In contrast, induced or constitutively resistant strains not only had reduced fitness but were severely impaired in colonization ability and dissemination among mice. These data indicate that tight regulation of resistance expression drastically reduces the biological cost associated with vancomycin resistance in Enterococcus spp. and accounts for the widespread dissemination of these strains. Our findings are in agreement with the observation that regulation of expression is common in horizontally acquired resistance and represents an efficient evolutionary pathway for resistance determinants to become selectively neutral.
Proceedings of the National Academy of Sciences 09/2010; 107(39):16964-9. · 9.68 Impact Factor
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ABSTRACT: Three Enterococcus faecium strains isolated successively from the same patient, vancomycin-resistant strain BM4659, vancomycin-dependent strain BM4660, and vancomycin-revertant strain BM4661, were indistinguishable by pulsed-field gel electrophoresis and harbored plasmid pIP846, which confers VanB-type resistance. The vancomycin dependence of strain BM4660 was due to mutation P(175)L, which suppressed the activity of the host Ddl D-Ala:D-Ala ligase. Reversion to resistance in strain BM4661 was due to a G-to-C transversion in the transcription terminator of the vanRS(B) operon that lowered the free energy of pairing from -13.08 to -6.65 kcal/mol, leading to low-level constitutive expression of the resistance genes from the P(RB) promoter, as indicated by analysis of peptidoglycan precursors and of VanX(B) D,D-dipeptidase activity. Transcription of the resistance genes, studied by Northern hybridization and reverse transcription, initiated from the P(YB) resistance promoter, was inducible in strains BM4659 and BM4660, whereas it started from the P(RB) regulatory promoter in strain BM4661, where it was superinducible. Strain BM4661 provides the first example of reversion to vancomycin resistance of a VanB-type dependent strain not due to a compensatory mutation in the ddl or vanS(B) gene. Instead, a mutation in the transcription terminator of the regulatory genes resulted in transcriptional readthrough of the resistance genes from the P(RB) promoter in the absence of vancomycin.
Antimicrobial Agents and Chemotherapy 04/2009; 53(5):1974-82. · 4.84 Impact Factor
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ABSTRACT: Since antibiotic resistance usually affords a gain of function, there is an associated biological cost resulting in a loss of fitness of the bacterial host. Considering that antibiotic resistance is most often only transiently advantageous to bacteria, an efficient and elegant way for them to escape the lethal action of drugs is the alteration of resistance gene expression. It appears that expression of bacterial resistance to antibiotics is frequently regulated, which indicates that modulation of gene expression probably reflects a good compromise between energy saving and adjustment to a rapidly evolving environment. Modulation of gene expression can occur at the transcriptional or translational level following mutations or the movement of mobile genetic elements and may involve induction by the antibiotic. In the latter case, the antibiotic can have a triple activity: as an antibacterial agent, as an inducer of resistance to itself, and as an inducer of the dissemination of resistance determinants. We will review certain mechanisms, all reversible, that bacteria have elaborated to achieve antibiotic resistance by the fine-tuning of the expression of genetic information.
Clinical Microbiology Reviews 02/2007; 20(1):79-114. · 16.13 Impact Factor
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ABSTRACT: The vanB operon of Enterococcus faecium BM4524 which confers inducible resistance to vancomycin is composed of the vanR(B)S(B) gene encoding a two-component regulatory system and the vanY(B)WH(B)BX(B) resistance genes that are transcribed from promoters P(RB) and P(YB) respectively. In this study, primer extension revealed transcription start sites at 13 and 48 bp upstream from the start codon of vanR(B) and vanY(B), respectively, that allowed identification of -10 and -35 promoter motifs. The VanR(B) protein was overproduced in Escherichia coli, purified and phosphorylated (VanR(B)-P) non-enzymically with acetylphosphate. VanR(B)-P and VanR(B) specifically bound to P(RB) and P(YB) promoters. VanR(B) bound at a single site at position -32.5 upstream from the P(RB) transcriptional start site and at two sites at positions -33.5 and -55.5 upstream from that of P(YB). The proximal VanR(B) binding site overlapped the -35 region of both promoters. VanR(B) was converted from a monomer to a dimer upon acetylphosphate treatment. VanR(B)-P had higher affinity than VanR(B) for its targets and appeared more efficient than VanR(B) in promoting open complex formation with P(RB) and P(YB). In the absence of regulator, E. coli RNA polymerase was able to interact with P(RB) but not with P(YB). Phosphorylation of VanR(B) significantly increased promoter interaction with RNA polymerase and led to an extended and modified footprint. In vitro transcription assays showed that VanR(B)-P activates P(YB) more strongly than P(RB). Analysis of the protected regions revealed one copy of a 21 bp sequence in the P(RB) promoter and two copies in the P(YB) promoter which may serve as recognition sites for VanR(B) and VanR(B)-P binding that are required for transcriptional activation and expression of vancomycin resistance.
Molecular Microbiology 08/2005; 57(2):550-64. · 5.01 Impact Factor
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ABSTRACT: A multiplex PCR assay was developed for detection of the six types of glycopeptide resistance characterized in enterococci and for identification of Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis at the species level. Primers targeting the genes vanA, vanB, vanC, vanD, vanE, vanG, and ddl of E. faecium and E. faecalis and nuc of S. aureus and a chromosomal portion specific to S. epidermidis were designed to allow amplification of fragments with various sizes. This specific and sensitive technique allows detection of glycopeptide-resistant strains, in particular methicillin-resistant S. aureus, that may escape phenotype-based automated rapid methods.
Journal of Clinical Microbiology 01/2005; 42(12):5857-60. · 4.15 Impact Factor
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ABSTRACT: Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.
Antimicrobial Agents and Chemotherapy 11/2004; 48(10):3892-904. · 4.84 Impact Factor
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ABSTRACT: VanB-type resistance in enterococci corresponds to resistance to vancomycin but not to resistance to the related glycopeptide teicoplanin, because the vanB gene cluster is activated by the VanR(B)-VanS(B) 2-component regulatory system in response to vancomycin but not to teicoplanin. Mutations in the vanS(B) gene allow for constitutive or teicoplanin-inducible expression of the resistance genes. To analyze in vivo expression of the van genes in rabbits with experimental endocarditis, a VanB-type Enterococcus faecalis with a transcriptional fusion between the P(YB) promoter of resistance genes and the gfpmut1 gene for the green-fluorescent protein in the chromosome was constructed. Rounded heaps containing fluorescent bacteria were detected in vegetation slides from rabbits treated with vancomycin but not in those from control rabbits, revealing induction of a tightly regulated vanB gene cluster. Teicoplanin-resistant mutants were detected as fluorescent bacteria in rabbits treated with teicoplanin. Thus, the reporter system monitored expression of a glycopeptide-resistance gene in vivo at a single-cell level.
The Journal of Infectious Diseases 02/2004; 189(1):90-7. · 6.41 Impact Factor
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ABSTRACT: Acquired VanG-type resistance to vancomycin (MIC = 16 micro g ml(-1)) but susceptibility to teicoplanin in Enterococcus faecalis BM4518 and WCH9 is due to the inducible synthesis of peptidoglycan precursors ending in d-alanine-d-serine. The vanG cluster, assigned to a chromosomal location, was composed of genes recruited from various van operons. The 3' end encoded VanG, a d-Ala:d-Ser ligase, VanXY(G), a putative bifunctional d,d-peptidase and VanT(G), a serine racemase: VanG and VanT(G) were implicated in the synthesis of d-Ala:d-Ser as in VanC- and VanE-type strains. Upstream from the structural genes for these proteins were vanW(G) with unknown function and vanY(G) containing a frameshift mutation which resulted in premature termination of the encoded protein and accounted for the lack of UDP-MurNAc-tetrapeptide in the cytoplasm. Without the frameshift mutation, VanY(G) had homology with Zn2+ dependent d,d-carboxypeptidases. The 5' end of the gene cluster contained three genes vanU(G), vanR(G) and vanS(G) encoding a putative regulatory system, which were co-transcribed constitutively from the PY(G) promoter, whereas transcription of vanY(G),W(G),G,XY(G),T(G) was inducible and initiated from the P(YG) promoter. Transfer of VanG-type glycopeptide resistance to E. faecalis JH2-2 was associated with the movement, from chromosome to chromosome, of genetic elements of c. 240 kb carrying also ermB-encoded erythromycin resistance. Sequence determination of the flanking regions of the vanG cluster in donor and transconjugants revealed the same 4 bp direct repeats and 22 bp imperfect inverted repeats that delineated the large element.
Molecular Microbiology 12/2003; 50(3):931-48. · 5.01 Impact Factor
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ABSTRACT: The consequences of VanD type glycopeptide resistance on the activity of vancomycin and teicoplanin were evaluated in vitro and in a rabbit model of aortic endocarditis with VanD type clinical isolate Enterococcus faecium BM4339 (MICs: vancomycin, 64 microg/ml; teicoplanin, 4 microg/ml) and its susceptible derivative BM4459 (MICs: vancomycin, 1 microg/ml; teicoplanin, 1 microg/ml). The two antibiotics were inactive against BM4339 in vivo, in terms both of reduction of bacterial counts and of prevention of emergence of glycopeptide-resistant subpopulations, despite using teicoplanin at concentrations greater than the MIC for VanD strains. This could be due to the high inoculum effect also observed in vitro with BM4339 and two other VanD type isolates against both antibiotics. These results suggest that detection of VanD type resistance is of major importance because it abolishes in vivo glycopeptide activity and allows the emergence of mutants highly resistant to glycopeptides.
Antimicrobial Agents and Chemotherapy 12/2003; 47(11):3515-8. · 4.84 Impact Factor
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ABSTRACT: Enterococcus faecium clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal vanB cluster, including the vanSB/vanRB two-component system regulatory genes. Enterococcus faecium strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The ddl gene of BM4525 had a 2 bp insertion leading to an impaired d-alanine:d-alanine ligase. Sequencing of the vanB operon in BM4525 also revealed an 18 bp deletion in the vanSB gene designated vanSBDelta. The resulting six amino acid deletion partially overlapped the G2 ATP-binding domain of the VanSBDelta histidine kinase leading to constitutive expression of the resistance genes. Sequence analysis indicated that the deletion occurred between two tandemly arranged heptanucleotide direct repeats, separated by 11 base-pairs. The VanSB, VanSBDelta and VanRB proteins were overproduced in Escherichia coli and purified. In vitro autophosphorylation of the VanSB and VanSBDelta histidine kinases and phosphotransfer to the VanRB response regulator did not differ significantly. However, VanSBDelta was deficient in VanRB phosphatase activity leading to accumulation of phosphorylated VanRB. Increased glycopeptide resistance in E. faecium BM4525 was therefore a result of the lack of production of d-alanyl-d-alanine ending pentapeptide and to constitutive synthesis of d-alanyl-d-lactate terminating peptidoglycan precursors, following loss of d-alanine:d-alanine ligase and of VanSB phosphatase activity respectively. We suggest that the heptanucleotide direct repeat in vanSB may favour the appearance of high level constitutively expressed vancomycin resistance through a 'slippage' type of genetic rearrangement in VanB-type strains.
Molecular Microbiology 12/2003; 50(3):1069-83. · 5.01 Impact Factor
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ABSTRACT: VanD type Enterococcus faecium 10/96A is constitutively resistant to vancomycin and to low levels of teicoplanin by nearly exclusive synthesis of peptidoglycan precursors terminating in D-alanyl-D-lactate (L. M. Dalla Costa, P. E. Reynolds, H. A. Souza, D. C. Souza, M. F. Palepou, and N. Woodford, Antimicrob. Agents Chemother. 44:3444-3446, 2000). A G(184)S mutation adjacent to the serine involved in the binding of D-Ala1 in the D-alanine:D-alanine ligase (Ddl) led to production of an impaired Ddl and accounts for the lack of D-alanyl-D-alanine-containing peptidoglycan precursors. The sequence of the vanD gene cluster revealed eight open reading frames. The organization of this operon, assigned to a chromosomal location, was similar to those in other VanD type strains. The distal part encoded the VanH(D) dehydrogenase, the VanD ligase, and the VanX(D) dipeptidase, which were homologous to the corresponding proteins in VanD-type strains. Upstream from the structural genes for these proteins was the vanY(D) gene; a frameshift mutation in this gene resulted in premature termination of the encoded protein and accounted for the lack of penicillin-susceptible D,D-carboxypeptidase activity. Analysis of the translated sequence downstream from the stop codon, but in a different reading frame because of the frameshift mutation, indicated homology with penicillin binding proteins (PBPs) with a high degree of identity with VanY(D) from VanD-type strains. The 5' end of the gene cluster contained the vanR(D)-vanS(D) genes for a putative two-component regulatory system. Insertion of ISEfa4 in the vanS(D) gene led to constitutive expression of vancomycin resistance. This new insertion belonged to the IS605 family and was composed of two open reading frames encoding putative transposases of two unrelated insertion sequence elements, IS200 and IS1341.
Antimicrobial Agents and Chemotherapy 02/2003; 47(1):7-18. · 4.84 Impact Factor
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ABSTRACT: Transposon Tn1546 confers resistance to glycopeptide antibiotics in enterococci and encodes two D,D-peptidases (VanX and VanY) in addition to the enzymes for the synthesis of D-alanyl-D-lactate (D-Ala-D-Lac). VanY was produced in the baculovirus expression system and purified as a proteolytic fragment that lacked the putative N-terminal membrane anchor of the protein. The enzyme was a Zn2+-dependent D,D-carboxypeptidase that cleaved the C-terminal residue of peptidoglycan precursors ending in R-D-Ala-D-Ala or R-D-Ala-D-Lac but not the dipeptide D-Ala-D-Ala. The specificity constants kcat/Km were 17- to 67-fold higher for substrates ending in the R-D-Ala-D-Ala target of glycopeptides. In Enterococcus faecalis, VanY was present in membrane and cytoplasmic fractions, produced UDP-MurNAc-tetrapeptide from cytoplasmic peptidoglycan precursors and was required for high-level glycopeptide resistance in a medium supplemented with D-Ala. The enzyme could not replace the VanX D,D-dipeptidase for the expression of glycopeptide resistance but a G237D substitution in the host D-Ala:D-Ala ligase restored resistance in a vanX null mutant. Deletion of the membrane anchor of VanY led to an active D,D-carboxypeptidase exclusively located in the cytoplasmic fraction that did not contribute to glycopeptide resistance in a D-Ala-containing medium. Thus, VanX and VanY had non-overlapping functions involving the hydrolysis of D-Ala-D-Ala and the removal of D-Ala from membrane-bound lipid intermediates respectively.
Molecular Microbiology 10/1998; 30(4):819 - 830. · 5.01 Impact Factor
