vanA and vanB incorporate into an endemic ampicillin-resistant vancomycin-sensitive Enterococcus faecium strain: effect on interpretation of clonality.
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Clonal spread and horizontal transfer in the spread of vancomycin resistance genes were investigated. Multiplex PCR, pulsed-field gel electrophoresis (PFGE), hybridization of enterococcal plasmids with the vanA and vanB probes, and sequencing of a fragment of vanB were used in the analysis. Before May 1996, 12 vancomycin-resistant Enterococcus faecium (VRE) isolates were found in Finland. Between May 1996 and October 1997, 156 VRE isolates were found in the Helsinki area. Between December 1997 and April 1998, fecal samples from 359 patients were cultured for VRE. One new case of colonization with VRE was found. During the outbreak period, 88% (137 of 155) of the VRE isolates belonged to two strains (VRE types I and II), as determined by PFGE. Each VRE type I isolate possessed vanB, and five isolates also had vanA. Of the 34 VRE type II isolates, 27 possessed vanA and 7 possessed vanB. Fifteen of 21 (71%) ampicillin-resistant, vancomycin-sensitive E. faecium (VSE) isolates found during and after the outbreak period in one ward were also of type II. Two VSE type II isolates were found in the hospital before the outbreak in 1995. By PFGE, the three groups (vanA, vanB, or no van gene) of type II shared the same band differences with the main type of VRE type II with vanA. None of the differences was specific to or determinative for any of the groups. Our material suggests that vanA and vanB incorporate into an endemic ampicillin-resistant VSE strain. (+info)
Physical mapping and functional assignment of the geranylgeranyl-bacteriochlorophyll reductase gene, bchP, of Rhodobacter sphaeroides.
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The bacteriochlorophyll of the purple photosynthetic bacterium Rhodobacter sphaeroides is esterified with phytol. The presence of this alcohol moiety is essential for the correct assembly of the photosynthetic apparatus. Despite this, and the fact that R. sphaeroides is widely used for the study of structure-function relationships in photosynthesis, the molecular genetics of the steps in which the alcohol is added and modified have not previously been investigated in this organism. Sequencing near the center of the photosynthesis gene cluster has now revealed the existence of an open reading frame encoding a putative 394-amino-acid polypeptide displaying strong homology with the products of a number of genes from other photosynthetic organisms, each proposed to be responsible for the reduction of the alcohol moiety of (bacterio)chlorophyll to phytol. An R. sphaeroides transposon mutant in this gene, bchP, possessed a structurally modified photosystem assembled with bacteriochlorophyll esterified with geranylgeraniol, rather than with phytol, implying that the product of this gene was geranylgeranyl-bacteriochlorophyll reductase. This identification was confirmed by the performance of in vitro assays using heterologously expressed protein, providing the first direct demonstration of the activity of a bchP gene product. (+info)
Prevalence and molecular epidemiology of glycopeptide-resistant enterococci in Belgian renal dialysis units.
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The molecular epidemiology of glycopeptide-resistant enterococci (GRE) colonizing the intestinal tracts of Belgian renal dialysis patients was studied among 1318 patients of a population of 1800 dialysis patients from 29 dialysis centers. Of these, 185 patients (14.0%) were colonized with a VANA-positive GRE; GRE harboring the VANB gene were not detected. The majority of the VANA GRE (80.5%) were identified as Enterococcus faecium; 14.8% were identified as E. faecalis; and a limited number were identified as E. avium, E. casseliflavus, E. dispar, E. durans, or E. gallinarum. Genome analysis of 277 VANA-positive GRE by pulsed-field gel electrophoresis revealed a high genetic variability both within the different dialysis centers and within the patients' own GRE flora. No high-level gentamicin-resistant VANA-positive GRE were detected, and most strains remained susceptible to ampicillin. These findings do not support a hospital-driven endemicity of VANA-positive enterococcal isolates in Belgium. (+info)
Substrate range and genetic analysis of Acinetobacter vanillate demethylase.
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An Acinetobacter sp. genetic screen was used to probe structure-function relationships in vanillate demethylase, a two-component monooxygenase. Mutants with null, leaky, and heat-sensitive phenotypes were isolated. Missense mutations tended to be clustered in specific regions, most of which make known contributions to catalytic activity. The vanillate analogs m-anisate, m-toluate, and 4-hydroxy-3,5-dimethylbenzoate are substrates of the enzyme and weakly inhibit the metabolism of vanillate by wild-type Acinetobacter bacteria. PCR mutagenesis of vanAB, followed by selection for strains unable to metabolize vanillate, yielded mutant organisms in which vanillate metabolism is more strongly inhibited by the vanillate analogs. Thus, the procedure opens for investigation amino acid residues that may contribute to the binding of either vanillate or its chemical analogs to wild-type and mutant vanillate demethylases. Selection of phenotypic revertants following PCR mutagenesis gave an indication of the extent to which amino acid substitutions can be tolerated at specified positions. In some cases, only true reversion to the original amino acid was observed. In other examples, a range of amino acid substitutions was tolerated. In one instance, phenotypic reversion failed to produce a protein with the original wild-type sequence. In this example, constraints favoring certain nucleotide substitutions appear to be imposed at the DNA level. (+info)
The biopesticide Paenibacillus popilliae has a vancomycin resistance gene cluster homologous to the enterococcal VanA vancomycin resistance gene cluster.
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We have previously identified, in Paenibacillus popilliae, a 708-bp sequence which has homology to the sequence of the enterococcal vanA gene. We have performed further studies revealing five genes encoding homologues of VanY, VanZ, VanH, VanA, and VanX in P. popilliae. The predicted amino acid sequences are similar to those in VanA vancomycin-resistant enterococci: 61% identity for VanY, 21% for VanZ, 74% for VanH, 77% for VanA, and 79% for VanX. The genes in P. popilliae may have been a precursor to or have had ancestral genes in common with vancomycin resistance genes in enterococci. The use of P. popilliae biopesticidal preparations in agricultural practice may have an impact on bacterial resistance in human pathogens. (+info)
Effects of the movement of insertion sequences on the structure of VanA glycopeptide resistance elements in Enterococcus faecium.
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A Tn1546-related element with IS1216V at position 8839 underwent a structural change after storage of the host strain of Enterococcus faecium at 4 degrees C. The element acquired IS1542 at position 3932, nucleotides 8732 to 8831 were deleted, and the first 3417 nucleotides were lost and replaced by an inverted copy of the IS1216V-vanY-vanZ-inverted-repeat block from the 3' end. Insertion sequence movement is likely to play a key role in the evolution of VanA resistance elements. (+info)
Characterization of vancomycin-resistant and vancomycin-susceptible Enterococcus faecium isolates from humans, chickens and pigs by RiboPrinting and pulsed-field gel electrophoresis.
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Forty-eight vancomycin-resistant and 35 vancomycin-sensitive Danish Enterococcus faecium isolates obtained from pigs, chickens and humans, as well as the human vanA reference isolate BM4147, were characterized by EcoRI RiboPrinting and SmaI pulsed-field gel electrophoresis. RiboPrinting of the 84 isolates yielded 40 types whereas PFGE-typing yielded 57 types discriminated by differences in more than three bands. By molecular typing, both clonal spread of E. faecium as well as horizontal transmission of Tn1546 between animals and humans was supported. Furthermore, it was found that the population of E. faecium spreads freely between the animal and human reservoir. (+info)
Enzymes of vancomycin resistance: the structure of D-alanine-D-lactate ligase of naturally resistant Leuconostoc mesenteroides.
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BACKGROUND: The bacterial cell wall and the enzymes that synthesize it are targets of glycopeptide antibiotics (vancomycins and teicoplanins) and beta-lactams (penicillins and cephalosporins). Biosynthesis of cell wall peptidoglycan requires a crosslinking of peptidyl moieties on adjacent glycan strands. The D-alanine-D-alanine transpeptidase, which catalyzes this crosslinking, is the target of beta-lactam antibiotics. Glycopeptides, in contrast, do not inhibit an enzyme, but bind directly to D-alanine-D-alanine and prevent subsequent crosslinking by the transpeptidase. Clinical resistance to vancomycin in enterococcal pathogens has been traced to altered ligases producing D-alanine-D-lactate rather than D-alanine-D-alanine. RESULTS: The structure of a D-alanine-D-lactate ligase has been determined by multiple anomalous dispersion (MAD) phasing to 2.4 A resolution. Co-crystallization of the Leuconostoc mesenteroides LmDdl2 ligase with ATP and a di-D-methylphosphinate produced ADP and a phosphinophosphate analog of the reaction intermediate of cell wall peptidoglycan biosynthesis. Comparison of this D-alanine-D-lactate ligase with the known structure of DdlB D-alanine-D-alanine ligase, a wild-type enzyme that does not provide vancomycin resistance, reveals alterations in the size and hydrophobicity of the site for D-lactate binding (subsite 2). A decrease was noted in the ability of the ligase to hydrogen bond a substrate molecule entering subsite 2. CONCLUSIONS: Structural differences at subsite 2 of the D-alanine-D-lactate ligase help explain a substrate specificity shift (D-alanine to D-lactate) leading to remodeled cell wall peptidoglycan and vancomycin resistance in Gram-positive pathogens. (+info)