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Antimicrobial Agents and Chemotherapy, May 2009, p. 1952-1963, Vol. 53, No. 5
0066-4804/09/$08.00+0 doi:10.1128/AAC.01348-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
New Combinations of Mutations in VanD-Type Vancomycin-Resistant Enterococcus faecium, Enterococcus faecalis, and Enterococcus avium Strains 
F. Depardieu,1
M.-L. Foucault,1
J. Bell,2
A. Dubouix,3
M. Guibert,4
J.-P. Lavigne,5
M. Levast,6 and
P. Courvalin1*
Unité des Agents Antibactériens, Institut Pasteur, 75724 Paris, Cedex 15,1 Department of Microbiology and Infection Control and Epidemiology and Public Health, Centre Hospitalier Universitaire Rangueil, Toulouse,3 Department of Internal Medicine, Hôpital Antoine Béclère, Clamart,4 INSERM, Espri 26, Université Montpellier 1, UFR de Médecine, 30908 Nimes Cedex,5 Service de Bactériologie, Centre Hospitalier de Chambéry, F-73011 Chambéry Cedex, France,6 Department of Infectious Diseases, Women's and Children's Hospital, North Adelaide, Australia2
Received 8 October 2008/ Returned for modification 27 January 2009/ Accepted 15 February 2009
We studied the clinical isolates Enterococcus faecium NEF1, resistant to high levels of vancomycin (MIC, 512 µg/ml) and teicoplanin (MIC, 64 µg/ml); Enterococcus faecium BM4653 and BM4656 and Enterococcus avium BM4655, resistant to moderate levels of vancomycin (MIC, 32 µg/ml) and to low levels of teicoplanin (MIC, 4 µg/ml); and Enterococcus faecalis BM4654, moderately resistant to vancomycin (MIC, 16 µg/ml) but susceptible to teicoplanin (MIC, 0.5 µg/ml). The strains were distinct, were constitutively resistant via the synthesis of peptidoglycan precursors ending in D-alanyl-D-lactate, and harbored a chromosomal vanD gene cluster that was not transferable. New mutations were found in conserved domains of VanSD: at T170I near the phosphorylation site in NEF1, at V67A at the membrane surface in BM4653, at G340S in the G2 ATP-binding domain in BM4655, in the F domain in BM4656 (a 6-bp insertion), and in the G1 and G2 domains of BM4654 (three mutations). The mutations resulted in constitutivity, presumably through the loss of the phosphatase activity of the sensor. The chromosomal Ddl D-Ala:D-Ala ligase had an IS19 copy in NEF1, a mutation in the serine (S185F) or near the arginine (T289P) involved in D-Ala1 binding in BM4653 or BM4655, respectively, and a mutation next to the lysine (P180S) involved in D-Ala2 binding in BM4654, leading to the production of an impaired enzyme. In BM4653 vanYD, a new insertion sequence, ISEfa9, belonging to the IS3 family, resulted in the absence of D,D-carboxypeptidase activity. Strain BM4656 had a functional D-Ala:D-Ala ligase, associated with high levels of both VanXD and VanYD activities, and is the first example of a VanD-type strain with a functional Ddl enzyme. Study of these five clinical isolates, displaying various assortments of mutations, confirms that all VanD-type strains isolated so far have undergone mutations in the vanSD or vanRD gene, leading to constitutive resistance, but that the Ddl host ligase is not always impaired. Based on sequence differences, the vanD gene clusters could be assigned to two subtypes: vanD-1 and vanD-4.
* Corresponding author. Mailing address: Unité des Agents Antibactériens, Institut Pasteur, 25, rue du Docteur 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
Published ahead of print on 2 March 2009.
Antimicrobial Agents and Chemotherapy, May 2009, p. 1952-1963, Vol. 53, No. 5
0066-4804/09/$08.00+0 doi:10.1128/AAC.01348-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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