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Antimicrobial Agents and Chemotherapy, January 1999, p. 41-47, Vol. 43, No. 1
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Vancomycin-Dependent Enterococcus faecalis Clinical Isolates and Revertant Mutants

Françoise Van Bambeke,1,dagger Murielle Chauvel,1 Peter E. Reynolds,2 Henry S. Fraimow,3 and Patrice Courvalin1,*

Unité des Agents Antibactériens, Institut Pasteur, 75724 Paris Cedex 15, France1; Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom2; and Department of Medicine, The Graduate Hospital, Philadelphia, Pennsylvania3

Received 24 June 1998/Returned for modification 25 August 1998/Accepted 13 October 1998

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.

* Corresponding author. Mailing address: Unité des Agents Antibactériens, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris Cedex 15, France. Phone: (33) (1) 45.68.83.20. Fax: (33) (1) 45.68.83.19. E-mail: pcourval{at}pasteur.fr.

dagger Present address: Unité de Pharmacologie Cellulaire et Moléculaire, Université Catholique de Louvain, 1200 Brussels, Belgium.

Antimicrobial Agents and Chemotherapy, January 1999, p. 41-47, Vol. 43, No. 1
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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Copyright © 1999 by the American Society for Microbiology. All rights reserved.