The mode of action of daptomycin against Staphylococcus aureus
Abstract number: 1732_164
Hobbs J., Miller K., Read N., Chopra I.
Objectives: The Silverman model for the mode of action of daptomycin (DAP) is based on the leakage of potassium ions (K+), membrane de-energisation and subsequent cell death without lysis. However, it is unknown whether loss of K+ is sufficient to cause cell death. In this study we determined the kinetics of both cell death and lysis of S. aureus treated with DAP to establish any relationships between the two.
Methods: Time-kill experiments were performed at various concentrations of DAP to determine the kinetics of cell death and samples were taken and prepared for scanning and transmission electron microscopy (EM). Culture samples were taken at intervals and the extracellular and intracellular amounts of ATP determined. The extracellular and intracellular levels of b-galactosidase (b-gal) in a S. aureus strain expressing b-gal in its cytoplasm were also measured.
Results: The time-kill experiments show DAP to be rapidly bactericidal at 32 mg/L. DAP at 4 mg/L shows a bacteriostatic effect for the first 60 min and then becomes slowly bactericidal. There is evidence for dose-dependent leakage of ATP and b-gal from cells treated with DAP, but this leakage occurs at very different rates. After 30 min of treatment with DAP at 32 mg/L, 90% of the total ATP has been released but only a negligible amount of b-gal. The percentage of b-gal released increases steadily over a period of hours, reaching 40% after 6 h and 90% after 24 h. After 30 min of treatment with DAP at 4 mg/L, 50% of the total ATP has been released and this amount continues to increase steadily. The leakage of b-gal is less apparent and after 24 h the cells have released less than 30%. The EM images show some cellular disruption in the first hour after DAP treatment, with this damage becoming more apparent and widespread over the following hours. The images clearly show that the rate and extent of cellular lysis is dose-dependent.
Conclusion: Our findings support the Silverman model for the mode of action of DAP and the hypothesis that it can cause rapid cell death without whole cell lysis. We have, however, shown that cell lysis does occur over time, through both EM images and the leakage of large amounts of b-gal. Our findings, while upholding the Silverman model, suggest that it is too simple to conclude that cell death is due only to loss of K+ and membrane de-energisation. The rapid loss of ATP would be an equally likely cause of cell death.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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