Vancomycin-dependent Enterococcus faecalis clinical isolates and revertant mutants.
Three vancomycin-dependent clinical isolates of Enterococcus faecalis of the VanB type were studied by determining (i) the sequence of the ddl gene encoding the host D-Ala:D-Ala ligase and the vanSB-vanRB genes specifying the two-component regulatory system that activates transcription of the vanB operon, (ii) the level of expression of resistance genes by using DD-dipeptidase activity as a reporter, and (iii) the proportions of the peptidoglycan precursors synthesized. Each strain had a mutation in ddl leading to an amino acid substitution (D295 to V; T316 to I) or deletion (DAK251-253 to E) at invariant positions in D-Ala:D-Ala, D-Ala:D-Lac, and D-Ala:D-Ser ligases. These mutations resulted in impaired host D-Ala:D-Ala ligases since only precursors terminating in D-Ala-D-Lac were synthesized under vancomycin-inducing conditions. Two types of vancomycin-independent revertants of one isolate were obtained in vitro after growth in the absence of vancomycin: (i) vancomycin-resistant, teicoplanin-susceptible mutants had a 6-bp insertion in the host ddl gene, causing the E251-to-EYK change that restored D-Ala:D-Ala ligase activity, (ii) constitutive vancomycin-resistant, teicoplanin-resistant mutants had substitutions (S232 to F or E247 to K) in the vicinity of the autophosphorylation site of the VanSB sensor and produced exclusively precursors ending in D-Ala-D-Lac. Vancomycin- and teicoplanin-dependent mutants obtained by growth in the presence of teicoplanin had an 18-bp deletion in VanSB, affecting residues 402 to 407 and overlapping the G2 ATP binding domain. The rapid emergence of vancomycin-independent revertants in vitro suggests that interruption of vancomycin therapy may not be sufficient to cure patients infected with vancomycin-dependent enterococci.
Van Bambeke
F
F
Unité des Agents Antibactériens, Institut Pasteur, 75724 Paris Cedex 15, France.
Chauvel
M
M
Reynolds
P E
PE
Fraimow
H S
HS
Courvalin
P
P
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial; D004591:Electrophoresis, Polyacrylamide Gel; D013293:Enterococcus faecalis / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D005838:Genotype; D009154:Mutation; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D017334:Teicoplanin / Q000494:pharmacology; D014640:Vancomycin / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; C079913:VanB protein, Enterococcus; D017334:Teicoplanin; D014640:Vancomycin; D010453:Peptide Synthases; C019638:D-alanylalanine synthetase