Figure 7.

Cholesteryl arachidonate and cholesteryl linoleate exhibit antibacterial activity against gram negative and gram positive bacteria in vitro. (a) Staining with the DNA probe Syto 9 as measurement for bacterial growth. P. aeruginosa were grown in trypticase soy broth, serially diluted, and Syto 9 fluorescence (RFU) and the number of colony forming units/ml were determined in parallel. Shown are means ± S.D., n = 4. In an MIC assay uninhibited bacteria reach concentrations of 10¹¹ CFU/ml. A reduction of RFU of 50% and 80% relates to a decrease in the number of colony forming units by 1 log and 1.5 log, respectively. (b) Dose dependent inhibitory activity of cholesteryl arachidonate (CA) and cholesteryl linoleate (CL) against P. aeruginosa. Bacteria were incubated in cation-adjusted Mueller-Hinton broth for 16 hours with and without liposomes containing cholesteryl esters (●) and vehicle liposomes (○) and relative fluorescence units were measured after addition of the DNA probe Syto 9. Shown are means + S.D., n = 3. (c) CFU assay with P. aeruginosa and the cholesteryl esters CA and CL. Bacteria were incubated for 3 hours in saline supplemented, low nutrient phosphate buffer in the presence or absence of vehicle and test liposomes and the number of CFU/ml was determined. Shown are means + S.D., n = 4 for CA and n = 3 for CL. *CFU/ml were significantly reduced to 20% + 16.99 of the inoculum, p = 0.002 in One Way ANOVA for Buffer vs CL. (d) Extended antibacterial activity testing of CA and CL in microtiter assay with Syto 9 read out. Cholesteryl esters were tested at 64 μg/ml; SE: Staphylococcus epidermidis, SA: Staphylococcus aureus, EF: Enterococcus faecalis, EC: Enterobacter cloacae. Data are expressed as % of RFU values for untreated bacteria. Shown are means + S.D., n = 3. *p < 0.001 in t-test for test liposomes compared to vehicle liposomes.