[Show abstract][Hide abstract] ABSTRACT: Enterococcus faecalis BM4518 is resistant to vancomycin by synthesis of peptidoglycan precursors ending in D-alanyl-D-serine. In the chromosomal vanG locus, transcription of the resistance genes from the PYG resistance promoter is inducible and, upstream from these genes, there is an unusual three-component regulatory system encoded by the vanURSG operon from the PUG regulatory promoter. In contrast to the other van operons in enterococci, the vanG operon possesses the additional vanUG gene which encodes a transcriptional regulator whose role remains unknown. We show by DNase I footprinting, RT-qPCR, and reporter proteins activities that VanUG, but not VanRG, binds to PUG and negatively autoregulates the vanURSG operon and that it also represses PYG where it overlaps with VanRG for binding. In clinical isolate BM4518, the transcription level of the resistance genes was dependent on vancomycin concentration whereas, in a ΔvanUG mutant, resistance was expressed at a maximum level even at low concentrations of the inducer. The binding competition between VanUG and VanRG on the PYG resistance promoter allowed rheostatic activation of the resistance operon depending likely on the level of VanRG phosphorylation by the VanSG sensor. In addition, there was cross-talk between VanSG and VanR'G, a VanRG homolog, encoded elsewhere in the chromosome indicating a sophisticated and subtle regulation of vancomycin resistance expression by a complex two-component system.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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. DOI:10.1073/pnas.1006855107 · 9.67 Impact Factor
[Show abstract][Hide abstract] 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 P175L, 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 vanRSB 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 PRB promoter, as indicated by analysis of peptidoglycan precursors and of VanXB d,d-dipeptidase activity. Transcription of the resistance genes, studied by Northern hybridization and reverse transcription,
initiated from the PYB resistance promoter, was inducible in strains BM4659 and BM4660, whereas it started from the PRB 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 vanSB gene. Instead, a mutation in the transcription terminator of the regulatory genes resulted in transcriptional readthrough
of the resistance genes from the PRB promoter in the absence of vancomycin.
[Show abstract][Hide abstract] ABSTRACT: We studied the clinical isolates Enterococcus faecium NEF1, resistant to high levels of vancomycin (MIC, 512 microg/ml) and teicoplanin (MIC, 64 microg/ml); Enterococcus faecium BM4653 and BM4656 and Enterococcus avium BM4655, resistant to moderate levels of vancomycin (MIC, 32 microg/ml) and to low levels of teicoplanin (MIC, 4 microg/ml); and Enterococcus faecalis BM4654, moderately resistant to vancomycin (MIC, 16 microg/ml) but susceptible to teicoplanin (MIC, 0.5 microg/ml). The strains were distinct, were constitutively resistant via the synthesis of peptidoglycan precursors ending in D-alanyl-D-lactate, and harbored a chromosomal vanD gene cluster that was not transferable. New mutations were found in conserved domains of VanS(D): at T(170)I near the phosphorylation site in NEF1, at V(67)A at the membrane surface in BM4653, at G(340)S in the G2 ATP-binding domain in BM4655, in the F domain in BM4656 (a 6-bp insertion), and in the G1 and G2 domains of BM4654 (three mutations). The mutations resulted in constitutivity, presumably through the loss of the phosphatase activity of the sensor. The chromosomal Ddl D-Ala:D-Ala ligase had an IS19 copy in NEF1, a mutation in the serine (S(185)F) or near the arginine (T(289)P) involved in D-Ala1 binding in BM4653 or BM4655, respectively, and a mutation next to the lysine (P(180)S) involved in D-Ala2 binding in BM4654, leading to the production of an impaired enzyme. In BM4653 vanY(D), a new insertion sequence, ISEfa9, belonging to the IS3 family, resulted in the absence of D,D-carboxypeptidase activity. Strain BM4656 had a functional D-Ala:D-Ala ligase, associated with high levels of both VanX(D) and VanY(D) activities, and is the first example of a VanD-type strain with a functional Ddl enzyme. Study of these five clinical isolates, displaying various assortments of mutations, confirms that all VanD-type strains isolated so far have undergone mutations in the vanS(D) or vanR(D) gene, leading to constitutive resistance, but that the Ddl host ligase is not always impaired. Based on sequence differences, the vanD gene clusters could be assigned to two subtypes: vanD-1 and vanD-4.
[Show abstract][Hide abstract] ABSTRACT: Background: Inducible expression of the vanB operon is controlled by the VanRBSB two-component regulatory system. Three VanB-type E. faecium were isolated successively from the same patient : inducibly Vm-resistant BM4659, Vm-dependent BM4660, and constitutively resistant BM4661. Methods: The strains were compared by PFGE and the ddl genes and vanB operons were sequenced. Peptidoglycan precursors from strains grown with or without Vm were separated by HPLC and the VanX D,D-dipeptidase and VanY D,D-carboxypeptidase activities were determined. Transcription of the vanB operon was studied by RT-PCR. Results: The strains had indistinguishable pulsotypes. Vm-dependence of BM4660 was due to mutation P175L in the host Ddl. The corresponding ddl gene and its RBS cloned under the control of a constitutive promoter did not confer Vm susceptibility to a VanD-type E. faecium with an impaired Ddl confirming that the Ddl in BM4660 was not functional. Constitutive resistance of BM4661 was due to a G to C mutation in the vanSB-vanYB intergenic region containing the resistance promoter region. This substitution lowered the pairing energy of the transcription terminator of vanSB from -13.08 to -6.65, leading to constitutive expression of the resistance genes based on quantitation of peptidoglycan precursors and of VanXB and VanYB activities. Transcription of the resistance genes was inducible in BM4659 and BM4660 and initiated from the resistance promoter whereas it was constitutive in BM4661 and initiated from the regulatory promoter. Conclusion: This is the first example of VanB-type constitutive glycopeptide resistance not due to combination of mutations in the ddl and vanSB genes. Instead, a mutation in the transcription terminator of the regulatory genes resulted in transcription of the resistance genes from the regulatory promoter in the absence of Vm.
Infectious Diseases Society of America 2008 Annual Meeting; 10/2008
[Show abstract][Hide abstract] ABSTRACT: Background: Conjugative transposon Tn1549 confers VanB-type inducible Vm resistance in enterococci. We have obtained transfer of Tn1549 from Clostridium symbiosum to enterococci in the absence of Vm. However, putative induction of transfer by Vm could not be assessed due to extremely low transfer frequency. Methods: Circular intermediates (CI) of the element and mRNA corresponding to the three functional modules (resistance, transposition, and conjugation) were quantified, respectively, by RT-PCR and real-time RT-PCR in cells grown with and without Vm. The effect of Orf7-8 on the Porf7 promoter was determined by quantification of CAT activity in Enterococcus faecalis JH2-2 harboring a chromosomal copy of orf7-8 constitutively expressed and a plasmid with a cat gene under the control of Porf7. Results: A 17-kb mRNA starting just downstream from the vanB operon and including orf7-8, xis, int, and orf16 to orf24 was detected, indicating transcriptional fusion through the joined ends of the CI, similarly to what has been observed with Tn916. Vm did not significantly alter the amount of CI. Vm increased transcription of the regulation and resistance operons with an induction ratio of ca. 5 fold for vanRB/SB, ca. 8,000 fold for vanB and vanYB, ca. 400 fold for orf7-8, ca. 100 fold for xis. In contrast, transcription of int and of the transfer module was not significantly altered. When constitutively expressed orf7-8 was provided in trans in E. faecalis JH2-2, the activity of Porf7 was increased, as detected by the CAT assay. This suggests that increased synthesis of Orf7-8 by Vm activated the Porf7 promoter which directs synthesis of the 17-kb mRNA. Conclusions: Vm increased the transcription of the transposition and resistance modules of Tn1549 with limited consequence on excision and conjugation of the element.
Infectious Diseases Society of America 2008 Annual Meeting; 10/2008
[Show abstract][Hide abstract] ABSTRACT: Background: VanD-type strains are constitutively resistant to Vm by synthesis of D-Ala-D-Lac ending peptidoglycan (PG) precursors due to mutations in the host D-Ala:D-Ala ligase and in the VanSD sensor. E. faecium NEF1, BM4653, BM4656 and E. avium BM4655 were isolates from France, and E. faecalis BM4654 from Australia. Methods: The strains were compared by PFGE and the ddl gene and vanD operon sequenced. PG precursors, VanX D,D-dipeptidase, and VanY D,D-carboxypeptidase activities were quantified. Results: The strains were distinct, constitutively resistant by synthesis of D-Ala-D-Lac ending precursors, and the vanD operons were similar. The Ddl had an IS19 copy in NEF1, a mutation in the serine (S185F) or near the arginine (T289P) involved in D-Ala1 binding in BM4653 and BM4655, respectively, or next to the lysine (P180S) involved in D-Ala2 binding in BM4654. The last 3 ddl genes cloned under the control of a constitutive promoter did not confer Vm susceptibility to a VanD-type E. faecium with an impaired Ddl confirming that their Ddls were not functional. The VanX and VanY activities, not required in the absence of D-Ala-D-Ala, were low. The Ddl of BM4656 was functional and associated with high VanX and VanY activities. Mutations in VanSD , T170I near the phosphorylation site in NEF1, V68A at the membrane surface in BM4653, in the G2 ATP-binding domain in BM4655, a 6-bp insertion in the F domain in BM4656 and 3 mutations in the G1 and G2 domains of BM4654 accounted for constitutivity. The mutant vanSD genes did not confer constitutive resistance to a VanD-type E. faecium with functional Ddl and impaired VanSD implying that the mutations led to loss of phosphatase activity. Conclusion: Four VanD-type strains had an impaired Ddl and were constitutively resistant due to a mutated VanSD. BM4656 was the first VanD-type strain with a functional Ddl and BM4655 the first VanD-type E. avium.
Infectious Diseases Society of America 2008 Annual Meeting; 10/2008
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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. DOI:10.1086/380566 · 6.00 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: Streptococcus pneumoniae clinical isolate BM4455 was resistant to 16-membered macrolides and to streptogramins. This unusual resistance phenotype
was due to an A2062C (Escherichia coli numbering) mutation in domain V of the four copies of 23S rRNA.
[Show abstract][Hide abstract] ABSTRACT: Resistance to macrolides in pneumococci is generally mediated by methylation of 23S rRNA via erm(B) methylase which can confer a macrolide (M)-, lincosamide (L)-, and streptogramin B (S(B))-resistant (MLS(B)) phenotype or by drug efflux via mef(A) which confers resistance to 14- and 15-membered macrolides only. We studied 20 strains with unusual ML or MS(B) phenotypes which did not harbor erm(B) or mef(A). The strains had been isolated from patients in Eastern Europe and North America from 1992 to 1998. These isolates were found to contain mutations in genes for either 23S rRNA or ribosomal proteins. Three strains from the United States with an ML phenotype, each representing a different clone, were characterized as having an A2059G (Escherichia coli numbering) change in three of the four 23S rRNA alleles. Susceptibility to macrolides and lincosamides decreased as the number of alleles in isogenic strains containing A2059G increased. Sixteen MS(B) strains from Eastern Europe were found to contain a 3-amino-acid substitution ((69)GTG(71) to TPS) in a highly conserved region of the ribosomal protein L4 ((63)KPWRQKGTGRAR(74)). These strains formed several distinct clonal types. The single MS(B) strain from Canada contained a 6-amino-acid L4 insertion ((69)GTGREKGTGRAR), which impacted growth rate and also conferred a 500-fold increase in MIC on the ketolide telithromycin. These macrolide resistance mechanisms from clinical isolates are similar to those recently described for laboratory-derived mutants.
[Show abstract][Hide abstract] ABSTRACT: Three of five natural plasmids carrying a wild-type vanA gene cluster did not confer LY333328 glycopeptide resistance on Enterococcus faecalis JH2-2 (MIC = 2 microg/ml). The two remaining plasmids conferred resistance to the drug (MIC, 8 microg/ml). The vanB gene cluster did not confer resistance to LY333328, since this antibiotic was not an inducer. Mutations in the vanS(B) sensor gene that allowed induction by teicoplanin or constitutive expression of the vanB cluster led to LY333328 resistance (MIC, 8 to 16 microg/ml). Overproduction of the VanH, VanA, and VanX proteins for D-alanyl-D-lactate (D-Ala-D-Lac) synthesis and D-Ala-D-Ala hydrolysis was sufficient for resistance to LY333328 (MIC, 16 microg/ml). Mutations in the host D-Ala:D-Ala ligase contributed to LY333328 resistance in certain VanA- and VanB-type strains, but the MICs of the antibiotic did not exceed 16 microg/ml. Addition of D-2-hydroxybutyrate in the culture medium of mutants that did not produce the VanH D-lactate dehydrogenase led to incorporation of this D-2-hydroxy acid at the C-terminal ends of the peptidoglycan precursors and to LY333328 resistance (MIC, 64 microg/ml). The vanZ gene of the vanA cluster conferred resistance to LY333328 (MIC, 8 microg/ml) by an unknown mechanism. These data indicate that VanA- and VanB-type enterococci may acquire moderate-level resistance to LY333328 (MIC </= 16 microg/ml) in a single step by various mechanisms.
Antimicrobial Agents and Chemotherapy 09/1999; 43(8):1875-80. · 4.48 Impact Factor