Disruption of biological membranes by hydrophobic molecules: a way to inhibit bacterial growth

Abstract

With antibiotic resistance increasing in the global population every year, efforts to discover new strategies against microbial diseases are urgently needed. One of the new therapeutic targets is the bacterial cell membrane since, in the event of a drastic alteration, it can cause cell death. We propose the utilization of hydrophobic molecules, namely, propofol (PFL) and cannabidiol (CBD), dissolved in nanodroplets of oil, to effectively strike the membrane of two well-known pathogens: Escherichia coli and Staphylococcus aureus. First, we carried out calorimetric measurements to evaluate the effects of these drugs on model membranes formed by lipids from these bacteria. We found that the drugs modify their transition temperature, enthalpy of cohesion, and cooperativity, which indicates a strong alteration of the membranes. Then, inhibition of colony-forming units is studied in incubation experiments. Finally, we demonstrate, using atomic force and fluorescence microscopy, that the drugs, especially propofol, produce a visible disruption in real bacterial membranes, explaining the observed inhibition. These findings may have useful implications in the global effort to discover new ways to effectively combat the growing threat of drug-resistant pathogens, especially in skin infections.

Keywords: CBD; E. coli; S. aureus; liposomes; propofol.

Figure 1

Characterization of the nanoemulsions through (A) size distribution, (B) kinetic stability, and (C) Zeta potential of the particles. The particle sizes and electric charge are very stable. Effects of nanoemulsions on liposomes using DSC: calorimetric profiles of E. coli (D) and S. aureus (E)-like-membranes. In both cases, PFL-OO 1% v/v has the greatest effect in the modification of cooperativity and transition temperature T_m with respect to the controls (black lines of profiles of pure liposomes) (see Table 1). Chemical structures of the evaluated drugs: propofol and CBD (F).

Figure 2

Schematic representation of the hydrophobic drug effects on the liposome model and bacterial membrane. The top panels depict a lipid vesicle before and after the drug insertion, and the lower panel schematizes a Gram-negative bacteria under such conditions.

Figure 3

Effect of various agents on E. coli and S. aureus bacteria with a final concentration of 24–10⁸ CFU/mL (Optical density of 0.04 A.U. at 600 nm). (A) For E. coli, we show that the vehicles (OO 1% v/v and OO 3% v/v) have the same small effect. Propofol-OO has a full bactericide effect, while CBD-OO needs to be augmented to 3% v/v to give rise to a 99% in the decrease of viability (2log reduction). In the vertical scale, –1 indicates that no bacteria survived after 10 h. (B) For S. aureus, the antimicrobial effectiveness of propofol is even better since –1 is obtained at 8 h. The minimum inhibitory concentration (MIC) of nanoemulsions (OO and PFL-OO) and PLF-DMSO was found in both bacteria after 12 h of cell culture growth (C, D). Three independent experiments were performed in our experiments. The median and the interquartile values in the error bars are shown. Asterisks mean the difference statistics tested with the Wilcoxon rank.

Figure 4

E. coli and S. aureus top images obtained by AFM. (A, E) with no treatment (controls), (B, F) with DMSO at 0.5%, and (C, G) with DMSO/PFL; E. coli and S. aureus, respectively. The images illustrate the dramatic damages caused in the cell membranes by PFL. Lengths and widths of the cells: for E. coli (D) and for S. aureus (H). In the case of E. coli, the histograms were obtained from 21 (no treatment), 43 (DMSO), and 27 (PFL) cells. For S. aureus, the histograms were calculated from 39 (no treatment), 25 (DMSO), and 25 (PFL) cells.

Figure 5

Contact-mode height AFM images of E. coli and S. aureus. (A, D) Show control cells, (B, E) after treatment with DMSO at 0.5%, and (C, F) after DMSO/PFL treatment, E. coli, and S. aureus, respectively. The first column corresponds to 3D topographic images, the second corresponds to 2D, and the third column corresponds to the height and size measurement of the selected cross-sectional line of the image. Black, green, and red lines are repetitions for three different bacteria. Despite the dispersion, it is clear the effect of PFL.

Figure 6

Fluorescence images of the effects of propofol on the bacterial membrane of E. coli and S. aureus cultures after 1.5 h treatment. The red fluorescence indicates that the phospholipid (TR-DHPE) penetrates the cell membranes. Clearly, in both cases, inward diffusion is promoted by the membrane disruption caused by propofol. Three groups are shown: control, drug carrier (DMSO), and drug (PFL). A 40X objective was used.