Bactericidal Activity of Sphingosine against A.a

We have previously shown that sphingosine has significant antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) isolates. However, there is little information available on potential antimicrobial activity against oral bacteria. Objectives: The purpose of this study was to determine if sphingosine exhibits antimicrobial activity against the periodontal pathogen Actinobacillus actinomycetemcomitans. Methods: Measurement of antimicrobial activity was achieved with a bactericidal kinetics assay approach. A.a. was cultured in BHIB in a 5% CO2 incubator at 37°C for 48 hours. Cells were harvested by centrifugation, resuspended in sterile ultra-pure water, and adjusted to achieve an assay concentration of 105 cfu/ml. Sphingosine was prepared to in ultra-pure water to obtain final assay concentrations ranging from 15 to 60 µg/ml in two-fold increments. Bactericidal kinetics assays were performed in small tubes. Aliquots of the assay tubes were removed over time and immediately diluted into Neutralizing Broth. Determination of the number of viable bacteria was done using an Autoplate 4000 spiral plating system. Raw bacterial counts were log10 transformed before statistical analysis. Results: Exposure of Aa to 60 µg/ml sphingosine resulted in a rapid killing of the cells, with a 2-order of magnitude decrease after one minute and complete killing by 2 minutes. This killing rate was equivalent to 0.1% chlorhexidine and not statistically different. Killing was also achieved at 15 µg/ml sphingosine, with a 2-order of magnitude decrease by 5 minutes of exposure. Complete killing was achieved by 10 minutes. Differences between the sphingosine and control were significantly different (p<0.05). Conclusion: These preliminary data suggest that significant bactericidal activity can be achieved with sphingosine against the periodontal pathogen A. a. Our ongoing studies are focusing on other periodontal pathogens and responses of polymicrobial suspensions and biofilms to sphingosine.