Serial daptomycin selection generates daptomycin-nonsusceptible Staphylococcus aureus strains with a heterogeneous vancomycin-intermediate phenotype

Abstract

In order to better understand the mechanism of daptomycin resistance, we generated a daptomycin-nonsusceptible derivative strain, strain 10*3d1 (MIC = 3.0 microg/ml), by in vitro exposure of methicillin-resistant Staphylococcus aureus strain N315DeltaIP (MIC = 0.5 microg/ml) to daptomycin. We also obtained a daptomycin-susceptible phenotypic revertant strain, strain 10*3d1-10 (MIC = 1.0 microg/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 N315DeltaIP were observed. Resistant strain 10*3d1 had the highest membrane potential, followed by strains 10*3d1-10 and N315DeltaIP. 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.

FIG. 1.

Growth curves of N315ΔIP and derivative strains. Circles, N315ΔIP; squares, 10*3d1; triangles 10*3d1-10.

FIG. 2.

Population analysis of N315ΔIP (open circles) and 10*3d1 (black squares). The numbers of colonies on plates containing various concentrations of daptomycin (A) and vancomycin (B) were counted after 72 h of incubation at 37°C due the slow growth of the nonsusceptible strain.

FIG. 3.

Membrane potential determination. The membrane potential increases with the fluorescence ratio (green/red). Gd, gadolinium (III) chloride (final concentration, 200 μM); 10*3d1 10th, 10*3d1-10.

FIG. 4.

Autolysis assay. Strain 209P (closed circles) was used as a control, since it has a high level of autolysin activity. Open circles, N315DIP; open squares, 10*3d1 10th; open triangles, 10*3d1-10.

FIG. 5.

Representative transmission electron micrographs and cell wall thicknesses of the strains studied. The values given under each panel are the means (nm) and standard deviations of the cell wall thickness from 30 cells of each strain determined at a magnification of ×25,000.