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Antimicrobial Agents and Chemotherapy, June 2009, p. 2312-2318, Vol. 53, No. 6
0066-4804/09/$08.00+0     doi:10.1128/AAC.01682-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Analysis of Cell Membrane Characteristics of In Vitro-Selected Daptomycin-Resistant Strains of Methicillin-Resistant Staphylococcus aureus{triangledown}

Nagendra N. Mishra,1 Soo-Jin Yang,1 Ayumi Sawa,1 Aileen Rubio,2 Cynthia C. Nast,3,4 Michael R. Yeaman,1,4 and Arnold S. Bayer1,4*

Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor—University of California at Los Angeles (UCLA) Medical Center, 1124 West Carson St., Torrance, California 90502,1 Cubist Pharmaceuticals, Lexington, Massachussetts,2 Cedars-Sinai Medical Center, Los Angeles, California,3 David Geffen School of Medicine at UCLA, Los Angeles, California4

Received 19 December 2008/ Returned for modification 15 February 2009/ Accepted 21 March 2009

Our previous studies of clinical daptomycin-resistant (Dapr) Staphylococcus aureus strains suggested that resistance is linked to the perturbations of several key cell membrane (CM) characteristics, including the CM order (fluidity), phospholipid content and asymmetry, and relative surface charge. In the present study, we examined the CM profiles of a well-known methicillin-resistant Staphylococcus aureus (MRSA) strain (MW2) after in vitro selection for DAP resistance by a 20-day serial passage in sublethal concentrations of DAP. Compared to levels for the parental strain, Dapr strains exhibited (i) decreased CM fluidity, (ii) the increased synthesis of total lysyl-phosphatidylglycerol (LPG), (iii) the increased flipping of LPG to the CM outer bilayer, and (iv) the increased expression of mprF, the gene responsible for the latter two phenotypes. In addition, we found that the expression of the dlt operon, which also increases positive surface charge, was enhanced in the Dapr mutants. These phenotypic and genotypic changes correlated with reduced DAP surface binding, mirroring observations made in clinical Dapr isolates. In this strain, serial exposure to DAP induced an increase in vancomycin MICs into the vancomycin-intermediate S. aureus (VISA) range (4 µg/ml) in parallel with increasing DAP MICs. Also, this Dapr strain exhibited significantly thicker cell walls than the parental strain, potentially correlating with the coevolution of the VISA phenotype and implicating cell wall structure and/or function in the Dapr phenotype. Importantly, despite the overexpression of mprF and dlt, the relative net positive surface charge was decreased in the Dapr mutants, suggesting that other factors contribute to the surface charge alterations and that a simple charge repulsion mechanism could not entirely explain the Dapr phenotype in these strains.

* Corresponding author. Mailing address: LA Biomedical Research Institute at Harbor-UCLA, 1124 West Carson Street, Bldg. RB2, Room 225, Torrance, CA 90502. Phone: (310) 222-6422. Fax: (310) 782-2016. E-mail: abayer{at}labiomed.org

{triangledown} Published ahead of print on 30 March 2009.

Antimicrobial Agents and Chemotherapy, June 2009, p. 2312-2318, Vol. 53, No. 6
0066-4804/09/$08.00+0     doi:10.1128/AAC.01682-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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