VanD-type vancomycin-resistant Enterococcus faecium and Enterococcus faecalis.
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.
Depardieu
Florence
F
Unité des Agents Antibactériens, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France. pcourval@pasteur.fr
Kolbert
Mathias
M
Pruul
Hendrik
H
Bell
Jan
J
Courvalin
Patrice
P
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology*; D002268:Carboxypeptidases / Q000235:genetics; D002876:Chromosomes, Bacterial / Q000235:genetics; D003198:Computer Simulation; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D004150:Dipeptidases / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D013293:Enterococcus faecalis / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D016984:Enterococcus faecium / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D010453:Peptide Synthases / Q000235:genetics* / Q000502:physiology*; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000235:genetics; D020713:Vancomycin Resistance / Q000235:genetics / Q000502:physiology*
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D002268:Carboxypeptidases; D004150:Dipeptidases; D010453:Peptide Synthases; C122181:VanD protein, Enterococcus faecium