Fig 1.
Synthesis of choline geranate.
CAGE was synthesized with equimolar molar equivalents of cholinium cation, geranate anion, and protonated geranic acid from choline bicarbonate and geranic acid.
Fig 2.
Cellular viability of MSSA biofilms vs time for exposures of neat CAGE (yellow), 0.1% CAGE (purple) and 10% bleach (green). Data is presented as calculated mean cellular viabilities ± standard deviation. A) Percent cellular viability for exposures on 24 hr biofilms, B) Percent cellular viability for exposures on 72 hr biofilms, C) Mean cellular viability vs exposure time for 24 hr biofilms, D) Mean cellular viability vs exposure time for 72 hr biofilms. Errors in a) and b) represent the standard error of the mean for n = 6, and statistical significance for all conditions compared to untreated controls were calculated via the Student’s t-test (* p<0.05, **p<0.01). Calculated means for data points in parts a and b vs time are presented in a logarithmic bar plot in parts c and d for the convenience of the reader. The lowest value on the y-axis of plots a and b represent the limit of detection of the implemented assay.
Fig 3.
SEM analysis of representative MSSA biofilms.
Biofilms were allowed to form onto titanium coupons for 72 hours and subsequently treated with 0.1% CAGE for a) 0, b) 5, and c) 120 minutes.
Fig 4.
SEM analysis of representative MSSA biofilms.
Biofilms were allowed to form onto titanium coupons for 72 hours and subsequently treated with 1% CAGE for a) 0, b) 5, and c) 60 minutes.
Table 1.
Observed minimal biofilm eradication concentrations (MBEC) for 30 and 120 minute exposures of dilute CAGE against 24 hour old biofilms of selected ESKAPE pathogens.
Each MBEC value represents the largest of three (n = 3) independent measurements.
Table 2.
Observed minimal biofilm eradication concentrations (MBEC) for 30 and 120 minute exposures of dilute CAGE against 72 hour old biofilms of selected ESKAPE pathogens.
Each MBEC value represents the largest of three (n = 3) independent measurements.
Fig 5.
Static representation of model POPE membranes in the presence of CAGE.
The lipid head groups are depicted in orange, the lipid tails in grey, cholinium cation in green, geranate in purple, and geranic acid in blue. Water molecules are omitted for clarity. Several periodic images are shown to make the long ranged structure clearer.
Table 3.
Effect of CAGE concentration on POPE area per lipid and bilayer thickness.
Fig 6.
Probability distribution of CAGE components into a model membrane.
Simulations defined the probability distribution of components perpendicular to the average bilayer surface with A) 8 CAGE, B) 16 CAGE, C) 32 CAGE, or D) 64 CAGE components added. The cholinium cation density is shown in green, geranate in blue, and geranic acid in purple. The zero point is the average position of the phosphorus atom from each lipid in the leaflet, where negative values are within the bilayer and positive values are in the aqueous phase. Results from each leaflet are averaged to improve statistics.
Fig 7.
CAGE migration over time within leaflets of the membrane bilayer.
Snapshots from the symmetric bilayer simulation with the CAGE components colored according to the leaflet they begin in. Panel A depicts an early production configuration, which shows geranate anions are highlighted in the top leaflet in blue and in the bottom leaflet in red and panel C shows the final configuration after 130 ns of simulation with anions all remaining within the leaflet that they begin. Similarly, panels B and D show geranic acid molecules at the beginning (panel B) and the end (panel D) after 130 ns of the production phase. The yellow and green molecules indicate molecules that begin in the upper (yellow) or lower (green) leaflets. The geranic acid is found to more freely traverse across the bilayer and randomize during our simulation as indicated by the yellow and green molecules randomizing at the end of the production phase.