SYNOPSIS Antimicrobial polypeptides including lysozymes (Ly) have membrane perturbing activity and are well documented effector molecules of innate immunity. In cystic fibrosis, a hereditary disease with frequent lung infection with Pseudomonas aeruginosa, the free fatty acid docosahexaenoic acid (DA), but not oleic acid (OA), is decreased and DA supplementation has been shown to improve the clinical condition in these patients. We hypothesized that DA may alone, or in conjunction with Ly, exert antibacterial action against P. aeruginosa. We found that DA and Ly synergistically inhibit the metabolic activity of P. aeruginosa, in contrast to OA. Electron microscopy and equilibrium dialysis suggest that DA accumulates in the bacterial membrane in the presence of Ly. Surface plasmon resonance with live bacteria and differential scanning calorimetry studies with bacterial model membranes reveal that, initially, DA facilitates lysozyme incorporation into the membrane, which in turn allows influx of more DA leading to bacterial cell death. Our study elucidates a molecular basis for the synergistic action of free fatty acids and antimicrobial polypeptides, which may be dysfunctional in cystic fibrosis.

BackgroundHealthcare associated infections (HAI) with multidrug-resistant (MDR) bacteria continue to be a global threat, highlighting an urgent need for novel antibiotics. In this study, we assessed the potential of free fatty acids and cholesteryl esters that form part of the innate host defense as novel antibacterial agents for use against MDR bacteria.MethodsLiposomes of six different phospholipid mixtures were employed as carrier for six different fatty acids and four different cholesteryl esters. Using a modified MIC assay based on DNA quantification with the fluoroprobe Syto9, formulations were tested against Gram-positive and Gram-negative bacteria implicated in HAI. Formulations with MIC values in the low μg/mL range were further subjected to determination of minimal bactericidal activity, hemolysis assay with sheep erythrocytes, and cytotoxicity testing with the human liver cell line HepG2. The potential for synergistic activity with a standard antibiotic was also probed.ResultsPalmitic acid and stearic acid prepared in carrier 4 (PA4 and SA4, respectively) were identified as most active lipids (MIC against MDR Staphylococcus epidermidis was 0.5 and 0.25 μg/mL, respectively; MIC against vancomycin resistant Enterococcus faecalis (VRE) was 2 and 0.5 μg/mL, respectively). Cholesteryl linoleate formulated with carrier 3 (CL3) exhibited activity against the S. epidermidis strain (MIC 1 μg/mL) and a Pseudomonas aeruginosa strain (MIC 8 μg/mL) and lowered the vancomycin MIC for VRE from 32–64 μg/mL to as low as 4 μg/mL. At 90 μg/mL PA4, SA4, and CL3 effected less than 5 % hemolysis over 3 h and PA4 and CL3 did not exhibit significant cytotoxic activity against HepG2 cells when applied at 100 μg/mL over 48 h.ConclusionsOur results showed that selected fatty acids and cholesteryl esters packaged with phospholipids exhibit antibacterial activity against Gram-positive and Gram-negative bacteria and may augment the activity of antibiotics. Bactericidal activity could be unlinked from hemolytic and cytotoxic activity and the type of phospholipid carrier greatly influenced the activity. Thus, fatty acids and cholesteryl esters packaged in liposomes may have potential as novel lipophilic antimicrobial agents.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-2138-8) contains supplementary material, which is available to authorized users.