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Inhibitory Effects of Sphinganine on Growth, Viability and Biofilm Formation in Staphylococcus aureus

K. Galbraith, M. Bankowski, L. R. Aaronson.

 

Certain lipids present in the skin inhibit the growth of gram-positive bacteria. Sphinganine (Sph), a precursor in the synthesis of complex sphingolipids, has antimicrobial properties contributing to the chemical barrier of skin. Previous studies have shown that increased concentrations of Sph resulted in reduced viability of Staphylococcus aureus. These studies showed that Sph caused the bacteria to become gram-negative and morphologically distorted. However, these studies added Sph in a concentration of ethanol that was itself inhibitory to the growth of S. aureus. It is unknown whether the Sph is creating the inhibitory effect or if the ethanol had a synergistic inhibitory effect. In the present studies, S. aureus was exposed to varying concentrations of Sph delivered to cultures in a final concentration of 1% ethanol, or in 0.1% Tergitol NP-40. In neither case did the vehicle alone inhibit growth of the bacteria, and Sph-induced loss of viability and changes in Gram staining characteristics were still observed at concentrations above 12.5 µM. Inhibition of growth and loss of viability under these conditions was reversible over time, as incubation with Sph beyond 6 hrs resulted in an increase in the number of colony-forming units in cultures. This suggests that S. aureus is able to overcome Sph inhibition, possibly by metabolizing the lipid into a nontoxic form. TEM of S. aureus cells exposed to Sph dissolved in ethanol showed uneven cell wall thickness and breaches in many cocci. Sph also had an inhibitory effect on the development of S. aureus biofilms. Dry mass of biofilms was reduced by approx. 50% in the presence of 50 µM Sph. Epifluorescence microscopy of Sph-treated biofilms labeled with Calcofluor white revealed a marked reduction in the abundance of extracellular polysaccharide (EPS), which was confirmed by the change in staining characteristics of colonies on Congo Red agar containing Sph. It is clear that concentrations of sphinganine below those found in skin cause inhibition of growth, reproduction, and biofilm production in S. aureus, and ongoing studies will attempt to elucidate the mechanisms of this inhibition.