Bactericidal Activity of Sphingosine against periodontal pathogens

Objectives: We have previously shown that sphingosine exerts significant bactericidal activity against Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, with complete killing of suspensions within 2-5 minutes of exposure. The purpose of this first phase of a two-phase study was to determine susceptibility of more putative periodontal pathogens and bacteria associated with health to sphingosine in monocultures. Methods: Fusobacterium nucleatum, Peptostreptococcus micros, Streptococcus sanguinis, S. oralis, and Actinomyces viscosus were used. A bactericidal kinetics approach was used to measure antimicrobial activity. Bacteria were cultured in their respective optimal media under anaerobic conditions. Sphingosine was prepared in sterile distilled water to obtain final assay concentrations ranging from 15-60 µg/ml in two fold increments. Assays were conducted with standardized bacterial suspensions prepared using predetermined standard curves. Aliquots were removed over time, diluted into Neutralizing Broth, and numbers of viable bacteria determined using spiral plating system. Raw bacterial counts were log10 transformed before repeated measures ANOVA was performed. Results: Exposure of F. nucleatum to 30 and 60 µg/ml sphingosine resulted in complete killing by 1 minute. P. micros exhibited complete killing at 2, 5 and 10 minutes exposed to 60, 30, and 15 µg/ml sphingosine respectively. Complete killing was not observed with A. viscosus, but differences between the sphingosine and control were significantly different (p<0.05) at 1, 2, and 5 minutes when exposed to 60, 30, and 15 µg/ml sphingosine respectively. Similar profiles were observed with the streptococci. Conclusion: These data confirm and expand our earlier findings that sphingosine exerts significant bactericidal activity against periodontal pathogens. Overall, the gram-positive bacteria exhibited much less killing upon exposure to sphingosine. Ongoing phase II studies are focusing on the response of polymicrobial suspensions and biofilms to sphingosine. Supported by T32DE0014678-06