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

In order to better understand the mechanism of daptomycin resistance, we generated a daptomycinnonsusceptible derivative strain, strain 103d1 (MIC 3.0 g/ml), by in vitro exposure of methicillinresistant Staphylococcus aureus strain N315IP (MIC 0.5 g/ml) to daptomycin. We also obtained a daptomycin-susceptible phenotypic revertant strain, strain 103d1-10 (MIC 1.0 g/ml), by passaging 103d1 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 103d1 and 103d1-10 compared to the membrane fluidity of N315IP were observed. Resistant strain 103d1 had the highest membrane potential, followed by strains 103d1-10 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 103d1 and 103d1-10. The vraF and vraG genes, which encode ATP binding cassette transporter proteins, were upregulated in 103d1. The vraSR two-component regulatory system was upregulated, and electron microscopy revealed that the cell wall of 103d1 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. After the isolation of the first methicillin-resistant Staphylococcus aureus (MRSA) strains with reduced susceptibility to vancomycin in 1997, the need for a drug with a new target increased (20, 21). Due to the multiple-drug resistance profile of most S. aureus strains isolated nowadays, there are few therapeutic options available. Daptomycin is a calcium-dependent cyclic lipopeptide approved by the U.S. Food and Drug Administration in 2003 and, afterwards, by the European Agency for Evaluation of Medicinal Products for the treatment of complicated skin and soft tissue infections caused by susceptible bacteria. The U.S. Food and Drug Administration also