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Antimicrobial Agents and Chemotherapy, December 2008, p. 4289-4299, Vol. 52, No. 12
0066-4804/08/$08.00+0     doi:10.1128/AAC.00417-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Serial Daptomycin Selection Generates Daptomycin-Nonsusceptible Staphylococcus aureus Strains with a Heterogeneous Vancomycin-Intermediate Phenotype

Ilana Lopes Baratella da Cunha Camargo , Hui-Min Neoh, Longzhu Cui, and Keiichi Hiramatsu^*

Department of Bacteriology, Juntendo University, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan

Received 28 March 2008/ Returned for modification 10 June 2008/ Accepted 19 September 2008

In order to better understand the mechanism of daptomycin resistance, we generated a daptomycin-nonsusceptible derivative strain, strain 10*3d1 (MIC = 3.0 µg/ml), by in vitro exposure of methicillin-resistant Staphylococcus aureus strain N315IP (MIC = 0.5 µg/ml) to daptomycin. We also obtained a daptomycin-susceptible phenotypic revertant strain, strain 10*3d1-10 (MIC = 1.0 µg/ml), by passaging 10*3d1 in drug-free medium for 10 days. The resultant triple-isogenic strains were analyzed for their phenotypes and gene expression by microarray analysis. No significant differences in the membrane fluidities of 10*3d1 and 10*3d1-10 compared to the membrane fluidity of N315IP were observed. Resistant strain 10*3d1 had the highest membrane potential, followed by strains 10*3d1-10 and N315IP. The vancomycin and teicoplanin MICs also increased. Teichoic acid genes (tagA, tagG), mprF encoding lysyl-phosphatidylglycerol, and cls encoding cardiolipin synthase were downregulated in 10*3d1 and 10*3d1-10. The vraF and vraG genes, which encode ATP binding cassette transporter proteins, were upregulated in 10*3d1. The vraSR two-component regulatory system was upregulated, and electron microscopy revealed that the cell wall of 10*3d1 was significantly thicker than that of the parental strain. Taken together, daptomycin exposure selected a daptomycin-nonsusceptible strain with a phenotype similar to that of heterogeneous vancomycin-intermediate S. aureus and a transcription profile that partially overlapped that of heterogeneous vancomycin-intermediate S. aureus.

Published ahead of print on 29 September 2008.

Present address: Universidade Federal de Minas Gerais, Belo Horizonte-MG, Brazil.

Present address: UKM Medical Molecular Biology Institute, Hospital Universiti Kebangsaan Malaysia, 56000 Cheras, Kuala Lumpur, Malaysia.