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Antimicrobial Agents and Chemotherapy, April 2005, p. 1419-1425, Vol. 49, No. 4
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.4.1419-1425.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Silencing of Glycopeptide Resistance in Enterococcus faecalis BM4405 by Novobiocin

Lorena Abadía Patiño,^1, Marc Chippaux,² Patrice Courvalin,^1* and Bruno Périchon¹

Unité des Agents Antibactériens, Institut Pasteur, Paris,¹ Laboratoire de Chimie Bactérienne, CNRS, Marseille, France²

Received 8 September 2004/ Returned for modification 23 October 2004/ Accepted 16 December 2004

Enterococcus faecalis BM4405-1, a susceptible derivative of the VanE-type vancomycin-resistant E. faecalis strain BM4405, was obtained after growth in the presence of novobiocin, an inhibitor of the GyrB subunit of DNA gyrase. In contrast to findings for BM4405, UDP-MurNAc-L-Ala--D-Glu-L-Lys-D-Ala-D-Ala (pentapeptide[D-Ala]) was the only peptidoglycan precursor found in BM4405-1, and no VanXY_E D,D-peptidase or VanT serine racemase activities were detected in that strain, even after induction by subinhibitory concentrations of vancomycin. Sequencing of the vanE operon of BM4405-1 revealed two mutations leading to substitutions in VanE (D200N) and in the C-terminal amino acid of VanR_E (Y225F). Cloning of the vanE, vanXY_E, and vanT_E genes of BM4405-1 into the susceptible E. faecalis strain JH2-2 conferred resistance to vancomycin, indicating that the mutation in vanE was not responsible for susceptibility. Transcriptional analysis of the vanE operon in BM4405 by quantitative reverse transcription-PCR indicated that novobiocin did not affect the expression level of the vanE operon. Sequencing of the gyrB gene of BM4405-1 revealed a mutation responsible for substitution of a residue (K337Y) required for ATPase activity and thus implicated in DNA supercoiling. Cloning of the gyrB gene of BM4405 restored vancomycin resistance to BM4405-1. Taken together, these data suggest that alteration of DNA supercoiling following a mutation in GyrB was responsible for lack of expression of the vanE operon and thus for vancomycin susceptibility in BM4405-1.

Present address: IIBCA Universidad de Oriente, Biomedica, Avenida Universidad, Cerro del Medio, 6101 Cumaná, Edo. Sucre, Venezuela.